Human Pellino polypeptides

ABSTRACT

There are disclosed novel polypeptides referred to as Pellino polypeptides, as well as fragments thereof, including immunogenic peptides. DNAs encoding such polypeptides as well as methods of using such DNAs and polypeptides are also disclosed.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/843,905, filed Apr. 27, 2001; which claims the benefit under35 U.S.C. 119(e) of U.S. provisional application Serial No. 60/200,198,filed Apr. 28, 2000; all of which are incorporated in their entirety byreference herein.

FIELD OF THE INVENTION

[0002] The invention is directed to molecules that are members of apolypeptide family referred to as Pellino (also called ConservedInflammatory Signal Target (CIST)). More particularly, the presentinvention includes Pellino polypeptides and fragments thereof, thenucleic acids encoding such polypeptides, and fragments thereof,processes for production of recombinant forms of such polypeptides,antibodies generated against these polypeptides, transgenic and knockoutcells and animals, and uses thereof.

BACKGROUND OF THE INVENTION

[0003] The Interleukin-1 (IL-1) pathway is a cellular signaling pathwayis that plays a crucial role in the mammalian inflammatory response.Several different receptors and ligands are involved in this pathway,including the ligands IL-1 alpha, IL-1 beta and IL-1 receptor antagonist(IL-1ra), and two IL-1 receptors referred to as IL-1 receptor Type I(IL-1RI) and IL-1 receptor Type II (IL-1RII); a soluble form of thelatter also exists. Of these, it appears that IL-1RI is the signalingreceptor, whereas IL-1RII does not transduce signal to a cell, butinstead may be involved in regulating an IL-1-mediated response (Colottaet al., Immunol. Today 15:562; 1994). Signaling via the IL-1 pathway iscomplex, requiring a number of accessory molecules in addition toIL-1RI, including a receptor-associated kinase (IRAK). Aserine/threonine kinase with homology to IRAK, referred to as Pelle, isfound in Drosophila (for review, see Belvin and Armstrong, Annu. Rev.Cell Dev. Biol. 12:393; 1996). Another Drosophila protein, Pellino, hasbeen reported to interact with Pelle (Grosshans et al., Mech. Dev.81:127; 1999).

[0004] Dorsal-ventral polarization in Drosophila embryos depends uponthe establishment of a gradient of nuclear localization of the Rel-liketranscription factor Dorsal. The transcriptional program mediated byDorsal results from a signaling cascade triggered by binding of anextracellular ligand Spaetzle to its receptor Toll. Intermediates ofthis signaling cascade include the adaptor protein Tube, theserine/threonine kinase Pelle, and Cactus, a cytosolic binding partnerof Dorsal. Signals transmitted by Toll result in the degradation ofCactus, and thereby permit the nuclear importation of Dorsal. Thesimilarity between the cytosolic domains of Toll and the mammalianinterleukin-1 receptor IL1-R1 was first noted by Gay and Keith (Gay, N.,and Keith, F., 1991, Nature 351: 355-356), and the number of proteinswhich contain the homologous regions, called the Toll/IL-1R (TIR) domainhas subsequently been extended to include a larger family of receptorsand intracellular signaling molecules from a variety of organisms. Thosewith leucine-rich repeats in their extracellular domains are broadlyinvolved in innate immune responses and include at least ten mammaliantoll-like receptors (TLRs) which initiate inflammatory responses tomicrobial pathogens such as peptidoglycan, bacterial lipopeptides,bacterial lipopolysaccharides, zymosan, CpG DNA, flagellin, lipoteichoicacids, and Respiratory Syncytial Virus proteins; and plant proteins suchas the N resistance gene product which mediate disease resistance.Furthermore, it is now clear that an important function of Tollsignaling in adult Drosophila is in controlling responses to fungalinfections.

[0005] Downstream components of the Toll signaling pathway have alsobeen evolutionarily conserved in mammalian TLR and interleukin-1receptor signaling pathways which culminate in nuclear translocation ofthe transcription factor Nuclear Factor kappa B (NF-kB). Protein kinasesof the IRAK family, close homologues of Pelle (such as IRAK and IRAK4),are recruited to the activated IL-1R or TLR receptor complexes throughthe adaptor protein MyD88 and undergo autophosphorylation reactions.Although MyD88 is not a strict analog of Tube, both proteins contain aso-called death domain, and Tube likely serves to mediate signaltransmission between Toll and Pelle, to which it binds. IRAKsubsequently interacts with another adaptor molecule TRAF6, which ishomologous to the recently described D-TRAF. Signals downstream of TRAF6appear to be divergent, and not all of them are fully understood, butone consequence, in mammalian cells, is the activation of the IkB kinase(IKK) complex which directly phosphorylates the inhibitory Cactushomolog IkB at two N-terminal serine residues causing its ubiquitinationand degradation. Released from a cytoplasmic association with IkB, NF-kBmigrates into the nucleus. Recently, a candidate for an additionalintermediate in Tube-Pelle interactions was found by yeast two-hybridscreening with Pelle as a bait sequence. This protein, called Pellino,was shown to interact with catalytically-competent Pelle, but not with amutant form of Pelle that lacked kinase activity. Although a functionfor Pellino was not addressed in this study, it was suggested that itcould either stabilize the activated form of Pelle, or mediate aninteraction with downstream Pelle substrates.

[0006] IL-1 and other pro-inflammatory cytokines have been implicated ina variety of diseases and conditions, including rheumatoid arthritis,multiple myeloma, osteoporosis, endotoxemia and sepsis, osteoarthritis,inflammatory bowel disease, and allergy. Inhibition of the signaling ofIL-1 using soluble forms of IL-1Rs, and the IL-1ra, have been shown tobe useful in treating or ameliorating disease characterized by excesslevels of IL-1 (Rosenwasser, J. Allergy Clin. Immunol. 102:344; 1998).Other parts of the IL-1 signaling pathway and other pro-inflammatory MAPkinase-activated pathways have also been the target of attempts toidentify additional molecules that can be used therapeutically tointervene in conditions related to IL-1 and pro-inflammatory cytokinesgenerally. Thus, there is a need in the art to identify novel moleculesinvolved in the IL-1 and MAP kinase-activated pro-inflammatory signalingpathways, both as tools with which to investigate cell signaling and foruse in identifying inhibitors of pro-inflammatory signaling. Ofparticular interest are novel polypeptides that are involved instimulation of multiple pro-inflammatory signaling pathways, asinhibition of such polypeptides would more effectively inhibitinflammatory effects than inhibition of a pathway-specific polypeptide.

SUMMARY OF THE INVENTION

[0007] The present invention is based upon the discovery of new murineand human Pellino polypeptides, murine Pellino-1 and -2, and humanPellino-1, -2, and -3.

[0008] The invention provides an isolated polypeptide capable ofstimulating MAP kinase-activated signaling pathways consisting of,consisting essentially of, or more preferably, comprising an amino acidsequence selected from the group consisting of:

[0009] (a) an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:12;

[0010] (b) an amino acid sequence selected from the group consisting of:amino acids ×1 to ×2 of SEQ ID NO:4, wherein ×1 is any of amino acids130 through 134 of SEQ ID NO:4, and ×2 is any of amino acids 187 through191 of SEQ ID NO:4; amino acids ×1 to ×2 of SEQ ID NO:8, wherein ×1 isany of the amino acids 132 through 136 of SEQ ID NO:8, and ×2 is any ofamino acids 189 through 193 of SEQ ID NO:8; and amino acids ×1 to ×2 ofSEQ ID NO:12, wherein ×1 is any of the amino acids 155 through 160 ofSEQ ID NO:12, and ×2 is any of amino acids 212 through 217 of SEQ ID NO:12;

[0011] (c) an amino acid sequence selected from the group consisting of:amino acids ×1 to ×2 of SEQ ID NO:4, wherein ×1 is any of amino acids 1through 10 of SEQ ID NO:4, and ×2 is any of amino acids 409 through 418of SEQ ID NO:4; amino acids ×1 to ×2 of SEQ ID NO:8, wherein ×1 is anyof amino acids 1 through 10 of SEQ ID NO:8, and ×2 is any of amino acids410 through 419 of SEQ ID NO:8; and amino acids ×1 to ×2 of SEQ IDNO:12, wherein ×1 is any of amino acids 1 through 10 of SEQ ID NO:12,and ×2 is any of amino acids 435 through 445 of SEQ ID NO: 12;

[0012] (d) an allelic variant of any of (a)-(c) above;

[0013] (e) a fragment of the amino acid sequences of any of (a)-(d)comprising at least 20 contiguous amino acids;

[0014] (f) a fragment of the amino acid sequences of any of (a)-(d),wherein a polypeptide consisting of said fragment is capable ofstimulating NF-kB-dependent or p38-dependent transcription;

[0015] (g) a fragment of the amino acid sequences of any of (a)-(d)comprising RING-finger-like domain amino acid sequences;

[0016] (h) an amino acid sequence comprising at least 20 amino acids andsharing amino acid identity with the amino acid sequences of any of(a)-(g), wherein the percent amino acid identity is selected from thegroup consisting of: at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%;

[0017] (i) an amino acid sequence of (h), wherein a polypeptidecomprising said amino acid sequence of (h) binds to an antibody thatalso binds to a polypeptide comprising an amino acid sequence of any of(a)-(g); and

[0018] (j) an amino acid sequence of (h) or (i) capable of stimulatingNF-kB-dependent or p38-dependent transcription.

[0019] Other aspects of the invention are isolated nucleic acidsencoding polypeptides of the invention, with a preferred embodimentbeing an isolated nucleic acid consisting of, or more preferably,comprising a nucleotide sequence selected from the group consisting of:

[0020] (a) SEQ ID NO:3;

[0021] (b) SEQ ID NO:7;

[0022] (c) SEQ ID NO:11;

[0023] (d) an allelic variant of (a)-(c);

[0024] (e) a nucleic acid, having a length of at least 15 nucleotides,that hybridizes under conditions of moderate stringency to the nucleicacid of any of claims (a) through (d);

[0025] (f) a nucleic acid comprising a nucleotide sequence that sharesnucleotide sequence identity with the nucleotide sequences of thenucleic acids of any of (a)-(e), wherein the percent nucleotide sequenceidentity is selected from the group consisting of: at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97.5%, at least 99%, and at least 99.5%.

[0026] The invention provides an isolated polypeptide capable ofinhibiting MAP kinase-activated signaling pathways consisting of,consisting essentially of, or more preferably, comprising an amino acidsequence selected from the group consisting of:

[0027] (a) an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:12, wherein amino acids 1through ×1 have been deleted from said sequence, and wherein ×1 is anyof amino acids 50 though 98 of said sequence;

[0028] (b) SEQ ID NO:4, wherein amino acids ×1 through ×2 have beendeleted from said sequence, and wherein ×1 is any amino acid from 99through 178 and ×2 is any amino acid from 100 through 179;

[0029] (c) SEQ ID NO:8, wherein amino acids ×1 through ×2 have beendeleted from said sequence, and wherein ×1 is any amino acid from 1through 180 and ×2 is any amino acid from 2 through 181;

[0030] (d) SEQ ID NO:12, wherein amino acids ×1 through ×2 have beendeleted from said sequence, and wherein ×1 is any amino acid from 1through 206 and ×2 is any amino acid from 2 through 207;

[0031] (e) an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:12, wherein one or more cysteineresidues of the RING-finger-like domain have been deleted or replaced bynon-cysteine residues;

[0032] (f) an allelic variant of (a)-(e);

[0033] (g) fragments of the amino acid sequences of any of (a)-(d) and(f) comprising RING-finger-like domain amino acid sequences;

[0034] (h) a fragment of the amino acid sequences of any of (a)-(g),wherein a polypeptide consisting of said fragment is capable ofinhibiting NF-kB -dependent or p38-dependent transcription;

[0035] (i) amino acid sequences comprising at least 20 amino acids andsharing amino acid identity with the amino-acid sequences of any of(a)-(h), wherein the percent amino acid identity is selected from thegroup consisting of: at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and atleast 99.5%;

[0036] (j) an amino acid sequence of (i), wherein a polypeptidecomprising said amino acid sequence of (i) binds to an antibody thatalso binds to a polypeptide comprising an amino acid sequence of any of(a)-(h); and

[0037] (k) an amino acid sequence of (i) or (j) capable of inhibitingNF-kB-dependent or p38-dependent transcription.

[0038] The invention also provides an isolated genomic nucleic acidcorresponding to the nucleic acids of the invention.

[0039] Other aspects of the invention are isolated nucleic acidsencoding polypeptides of the invention, allelic variants of thesenucleic acids, and isolated nucleic acids, preferably having a length ofat least 15 nucleotides, that hybridize under conditions of moderatestringency to the nucleic acids encoding polypeptides of the invention.In preferred embodiments of the invention, such nucleic acids encode apolypeptide having Pellino polypeptide activity or Pellinodominant-negative activity, or comprise a nucleotide sequence thatshares nucleotide sequence identity with the nucleotide sequences of thenucleic acids of the invention, wherein the percent nucleotide sequenceidentity is selected from the group consisting of: at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97.5%, at least 99%, and at least 99.5%.

[0040] Further provided by the invention are expression vectors andrecombinant host cells comprising at least one nucleic acid of theinvention, and preferred recombinant host cells wherein said nucleicacid is integrated into the host cell genome.

[0041] Also provided is a process, for producing a polypeptide encodedby the nucleic acids of the invention, comprising culturing arecombinant host cell under conditions promoting expression of saidpolypeptide, wherein the recombinant host cell comprises at least onenucleic acid of the invention. A preferred process provided by theinvention further comprises purifying said polypeptide. In anotheraspect of the invention, the polypeptide produced by said process isprovided.

[0042] Further aspects of the invention are isolated antibodies thatbind to the polypeptides of the invention, preferably monoclonalantibodies, also preferably humanized antibodies or humanizedantibodies, and preferably wherein the antibody inhibits the activity ofsaid polypeptides.

[0043] The invention additionally provides a method of designing aninhibitor of the polypeptides of the invention, the method comprisingthe steps of determining the three-dimensional structure of any suchpolypeptide, analyzing the three-dimensional structure for the likelybinding sites of substrates, synthesizing a molecule that incorporates apredicted reactive site, and determining the polypeptide-inhibitingactivity of the molecule.

[0044] In a further aspect of the invention, methods are provided foridentifying compounds that alter Pellino polypeptide activity (orPellino dominant-negative activity) comprising

[0045] (a) mixing a test compound with a polypeptide of the invention;and

[0046] (b) determining whether the test compound alters the Pellinopolypeptide activity (or Pellino dominant-negative activity) of saidpolypeptide.

[0047] In another aspect of the invention, a method is providedidentifying compounds that inhibit the binding activity of Pellinopolypeptides comprising

[0048] (a) mixing a test compound with a polypeptide of the inventionand a binding partner of said polypeptide; and

[0049] (b) determining whether the test compound inhibits the bindingactivity of said polypeptide.

[0050] In preferred embodiments, the binding partner is an intracellularsignaling pathway molecule; more preferably, the binding partner isselected from the group consisting of TRAF6, IRAK, and IRAK4.

[0051] In another aspect of the invention, a method is provided foridentifying peptide agonists and antagonists of the Pellino polypeptidesof the invention, the method comprising selecting at least one peptidethat binds to a polypeptide of the invention, wherein the peptide isselected in a process comprising one or more techniques selected fromyeast-based screening, rational design, protein structural analysis,screening of a phage display library, an E. coli display library, aribosomal library, an RNA-peptide library, and a chemical peptidelibrary. In further aspects of the invention, the peptide is selectedfrom a plurality of randomized peptides.

[0052] The invention also provides methods for stimulatingNF-kB-dependent or p38-dependent transcription, or for stimulating acellular response to an intercellular signal molecule, comprisingproviding at least one compound selected from the group consisting ofthe polypeptides of the invention and agonists of said polypeptides;with a preferred embodiment of the method further comprising increasingsaid activities in a patient by administering at least one polypeptideof the invention. Preferably, the intercellular signal molecule isselected from the group consisting of interleukin-1 (IL-1), TNF-alpha,IL-18, phorbol 12-myristate 13-acetate (PMA), peptidoglycan, bacteriallipopeptides, bacterial lipopolysaccharides, zymosan, CpG DNA,flagellin, lipoteichoic acids, and Respiratory Syncytial Virus proteins.Also preferably, the cellular response is translocation of NF-kB to thecell nucleus, an increase in NF-kB-dependent transcription, or anincrease in p38-dependent transcription.

[0053] Further provided by the invention is a method for inhibitingNF-kB-dependent or p38-dependent transcription, comprising providing atleast one antagonist of the polypeptides of the invention; with apreferred embodiment of the method further comprising decreasing saidactivities in a patient by administering at least one antagonist of thepolypeptides of the invention, and with a further preferred embodimentwherein the antagonist is an antibody that inhibits the activity of anyof said polypeptides.

[0054] The invention additionally provides methods for preventing ortreating infection by a pathogen, or for inhibiting apoptosis,comprising administering at least one compound selected from the groupconsisting of the polypeptides of the invention and agonists of saidpolypeptides; with a preferred embodiment wherein the pathogen isselected from the group consisting of prions, viruses, bacteria, fungi,algae, and protozoa.

[0055] In other aspects of the invention, methods are provided fortreating cancer or an inflammatory condition comprising administering anantagonist of wild-type Pellino polypeptides of the invention; with apreferred embodiment wherein the inflammatory condition is selected fromthe group consisting of asthma, rheumatoid arthritis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, atherosclerosis, andAlzheimer's disease.

[0056] A further embodiment of the invention provides a use for the“dominant-negative” Pellino polypeptides of the invention in thepreparation of a medicament for treating an inflammatory condition; witha preferred embodiment wherein the inflammatory condition is selectedfrom the group consisting of asthma, rheumatoid arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, atherosclerosis, andAlzheimer's disease.

[0057] Also comprehended within the scope of the instant invention arefusion proteins comprising any of the aforementioned polypeptides and apolypeptide selected from the group consisting of an immunoglobulin Fcdomain, a FLAG peptide, a peptide comprising at least about 6 Hisresidues, a leucine zipper, a GFP peptide, a PkA peptide, a birApeptide, and a GST peptide. Nucleic acid molecules that encode suchfusion proteins are also included within the instant invention, as arerecombinant expression vectors comprising any of the aforementionedDNAs, host cells transformed or transfected with such expressionvectors, and processes for preparing polypeptides, comprising culturingsuch host cells under conditions promoting expression, and recoveringthe polypeptides. The invention further provides transgenic or knockoutanimals generated by using the inventive DNAs.. The invention furtherprovides antibodies that specifically binds the inventive polypeptides,including monoclonal antibodies and human antibodies. Assays foridentification of small molecules that regulate IL-1 signaling,utilizing an inventive peptide, are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1. This schematic diagram shows our model for theIL-1-mediated signaling pathway involving Pellino-1.

[0059]FIG. 2. Panels A and B show IL-1-induced interaction of Pellino-1with IL-1 signaling components. Cell extracts from 293 cells (293, PanelA) or 293 cells transfected with flag-Pellino-1 (293-Pellino-1, Panels Aand B), either untreated (0) or stimulated with IL-1 for indicatedtimes, were immuno-precipitated with the anti-flag antibody M2 (Panels Aand B), anti-TRAF6 (Panel B), and a control antibody (anti-p27, 1h*,Panel A), followed by Western analyses with antibodies against thefollowing: the flag-tag on Pellino-1 (Panels A and B), IRAK (Panel A),IRAK4 (Panel A), TRAF6 (Panels A and B), IL-1R (Panel B), TAK1 (PanelB), and TAB2 Panel B).

[0060]FIG. 3. Experiments indicating that reduced expression ofPellino-1 impairs IL-1 signaling. Panel A shows the identification ofclones with reduced expression of Pellino-1. Total RNA samples from 293cells stably transfected with Pellino-1-pSUPER, which generatessiRNA-like molecules directed against Pellino-1 RNA, were analyzed byNorthern procedure with a Pellino-1 gene-specific probe. ‘C’ indicatesnon-transfected cells; cells corresponding to the lanes marked with anasterisk were selected for further analysis: lane 9 cells (C-9) appearto have unaltered Pellino-1 expression, while cells corresponding tolanes 12 and 16 (C-12 and C-16, respectively) appear to have reducedPellino-1 expression. Panel B shows the results of an experiment inwhich cell extracts from clones C-9, C-12, and C-16, either untreated orstimulated with IL-1 (2, 4 and 8 h) or with TNF (TNF), were analyzed byan NF-kB gel shift assay as described in Example 8. IL-1 induced muchweaker NF-kB activation in C-12 and in C-16 than in C-9, suggesting thatPellino-1 is required for activation of NF-kB by IL-1. Panel C shows RNAsamples from C-9, C-12, and C16 cells, either untreated (0) orstimulated with IL-1 (4, 8, 12, 24 h), analyzed on a Northern bothprocedure using IL-8 and GAPDH cDNAs as probes. IL-1-induced IL-8 geneexpression in C-12 and C-16 cells was greatly reduced as compared to itsexpression in C-9 cells, indicating that Pellino-1 is also required forinduction of IL-8 gene expression by IL-1.

DETAILED DESCRIPTION OF THE INVENTION

[0061] We have identified murine and human Pellino-1 and -2, and humanPellino-3, new Pellino polypeptides having structural featurescharacteristic of the Pellino polypeptide family. By expression of oneof these mammalian Pellino isoforms in COS cells, murine Pellino-1, weshow that various inducers of NF-kB specifically cause Pellino-1 to beproteolytically processed into an insoluble form. Furthermore, wedemonstrate that expression of Pellino polypeptides such as Pellino-1and Pellino-2 strongly activates NF-kB-dependent reporter genes andaugments Jun N-terminal kinase, p38 kinase, and ERK signaling mediatedby IL-1, and that mutant forms of Pellino, lacking conserved motifs,suppress basal and cytokine-induced NF-kB activation, and alsop38-dependent transcription. The molecules of this invention haveutility as, or lead to, anti-inflammatory therapies. The discovery ofthe polynucleotides of the invention enables the construction ofexpression vectors comprising DNA that encodes polypeptides; host cellstransfected or transformed with the expression vectors; development oftransgenic and knockout cells and animals; isolated and purifiedpolypeptides and fragments thereof; the use of the polynucleotidesthereof as probes or primers to identify DNA encoding proteins havingPellino activity, the use of single-stranded sense or antisenseoligonucleotides from the nucleic acids to inhibit expression and/orfunction of polynucleotide encoded by the Pellino genes; the use of suchpolynucleotides or polypeptides to identify small molecule inhibitors ofprotein association or function of Pellino; the use of suchpolynucleotides or polypeptides to identify other molecules involved inIL-1 signaling; the use of such polypeptides and fragments thereof togenerate antibodies; and the use of such antibodies to purify thePellino polypeptide.

[0062] The amino acid sequences of murine and human Pellino-1, murineand human Pellino-2, and human Pellino-3 polypeptide are provided in SEQID NOs 2, 4, 6, 8, and 12, respectively, and an alignment showing thesequence similarities between murine and human Pellino-1 and -2, humanPellino-3, and other Pellino polypeptides is presented in Table 1 inExample 1 below. The Pellino polypeptide family is remarkably wellconserved, with the human family members highly similar to each other,and extremely similar to homologous Pellino family members from otherspecies such as Mus musculus.

[0063] Typical structural elements common to members of the Pellinopolypeptide family include a particularly well-conserved central domain,extending from amino acid 132 through amino acid 193 in Pellino-1 (SEQID NOs 2 and 4; which corresponds to amino acids 134 through 195 in SEQID NO:8 and amino acids 158 through 219 in SEQ ID NO:12); an absolutelyconserved motif from residue 245 through residue 254 of SEQ ID NOs 2 and4 (which corresponds to amino acids 247 through 256 in SEQ ID NO:8 andamino acids 271 through 280 in SEQ ID NO:12); and a domain (“theRING-finger-like domain”), similar to the C3HC4 RING-finger subfamily ofZinc-finger domains, from amino acid 333 through amino acid 398 of SEQID NOs 2 and 4 (which corresponds to amino acids 335 through 400 in SEQID NO:8 and amino acids 360 through 425 in SEQ ID NO:12). There arecertain key cysteine residues within the RING-finger-like domain, suchthat substitutions of those residues are likely be associated with analtered function or lack of that function for Pellino polypeptides. Theconserved cysteine residues within the Pellino polypeptides are locatedat positions 333, 336, 367, 371, 395, and 398 of SEQ ID NOs 2 and 4 (andat positions 335, 338, 369, 373, 397, and 400 of SEQ ID NO:8 and thecorresponding positions in SEQ ID NO:6, and at positions 360, 363, 394,398, 422, and 425 of SEQ ID NO:12). The skilled artisan will recognizethat the boundaries of the regions of murine and human Pellino-1 and -2,and human Pellino-3 polypeptides described above are approximate andthat the precise boundaries of such domains can also differ from memberto member within the Pellino polypeptide family. However, it is clearfrom the above and from Table 1 that murine and human Pellino-1 and -2and human Pellino-3 polypeptides each have an overall structureconsistent with each other and with other Pellino polypeptides.

[0064] Biological activities or functions associated with murine andhuman Pellino-1 and -2, and human Pellino-3 polypeptides, includestimulation of MAP kinase-activated signaling pathways, such aspro-inflammatory signaling pathways, and in particular stimulation oftranscription from downstream promoters such as NF-kB- and p38-dependentpromoters. The ability of murine and human Pellino-1 and -2 and humanPellino-3 polypeptides to stimulate MAP kinase-activated signalingpathways is associated with many domains of the Pellino polypeptides(such as the N-terminal, central conserved domain, the RING-finger-likedomain, and the C-terminal domain) or with the polypeptides in theirentirety, as deletions of N- or C-terminal domains and certainmodifications of key residues within murine Pellino-I have been showneither to abolish this stimulatory activity, or to generate “dominantnegative” Pellino-1 mutants which inhibit MAP kinase-activated signalingpathways. The ability of murine and human Pellino-1 and -2 and humanPellino-3 polypeptides to stimulate MAP kinase-activated signalingpathways can be determined, for example, in an assay that measures thetranscription of reporter genes, such as the luciferase coding sequenceor the chloramphenicol acetyltransferase (CAT) coding sequence, fromdownstream promoters, cush as the NF-kB-dependent IL-8 promoter or thep38-dependent CHOP promoter. Pellino polypeptides that stimulate MAPkinase-activated signaling pathways preferably have at least 10% (morepreferably, at least 25%, and most preferably, at least 50%) of thisstimulatory activity as compared to that of murine Pellino-1 measured inthe NF-kB-dependent IL-8 promoter-luciferase reporter gene assays ofExample 2.

[0065] Murine and human Pellino-1 and -2, and human Pellino-3polypeptides, are also substrates for proteases, such aschymotrypsin-like serine proteases, and demonstrate a change insolubility in response to stimulation of cells by stimulatory moleculessuch as TNF-alpha and PMA. The protease-substrate activity is associatedwith the central domain of murine and human Pellino-1 and -2 and humanPellino-3 polypeptides, this central domain comprising residues 154 and165 of SEQ ID NO:2 (or the corresponding residues of other Pellinopolypeptides), substitutions to which have been shown to reduce thecleavage of Pellino-1. Thus, for uses requiring Pellinoprotease-substrate activity, preferred murine and human Pellino-1 and -2and human Pellino-3 polypeptides include those comprising residues 154and 165 of SEQ ID NO:2 (or the corresponding residues of other Pellinopolypeptides) or having the conserved central domain, and exhibitingproteolytic cleavage in response to appropriate cell stimuli, such astreatment with TNF-alpha or PMA. Preferred murine and human Pellino-1and -2 and human Pellino-3 polypeptides further include oligomers orfusion polypeptides comprising at least one conserved central domain ofone or more murine and human Pellino-1 and -2 and human Pellino-3polypeptides, and fragments of any of these polypeptides, exhibitingproteolytic cleavage in response to appropriate cell stimuli, such astreatment with TNF-alpha or PMA. The protease-substrate activity ofmurine and human Pellino-1 and -2 and human Pellino-3 polypeptides canbe determined, for example, in an assay that measures the extent ofPellino polypeptide cleavage as described in Examples 3 and 4 below.Pellino polypeptides having protease-substrate activity preferably haveat least 10% (more preferably, at least 25%, and most preferably, atleast 50%) of the protease-substrate activity of murine Pellino-1-FLAGas measured in the assays of Examples 3 and 4.

[0066] Biological activities or functions associated with certain mutantor altered forms of murine and human Pellino-1 and -2, and humanPellino-3 polypeptides, include inhibition of MAP kinase-activatedsignaling pathways, such as pro-inflammatory signaling pathways, and inparticular inhibition of transcription from downstream promoters such asNF-kB- and p38-dependent promoters. The ability of these mutant murineand human Pellino-1 and -2 and human Pellino-3 polypeptides to inhibitMAP kinase-activated signaling pathways is associated with alterationsto certain domains of the Pellino polypeptides such as the N-terminalregion, central conserved domain, and the RING-finger-like domain, asdeletions of 50 or 99 N-terminal amino acids and certain modificationsto the central conserved domain or the RING-finger-like domain withinmurine Pellino-1 have been shown to generate “dominant negative”Pellino-1 mutants which inhibit MAP kinase-activated signaling pathways(see Example 2, below). The ability of altered murine and humanPellino-1 and -2 and human Pellino-3 polypeptides to inhibit MAPkinase-activated signaling pathways can be determined, for example, inan assay that measures the transcription of reporter genes, such as theluciferase coding sequence or the chloramphenicol acetyltransferase(CAT) coding sequence, from downstream promoters, such as theNF-kB-dependent IL-8 promoter or the p38-dependent CHOP promoter.Pellino polypeptides that inhibit MAP kinase-activated signalingpathways preferably have at least 10% (more preferably, at least 25%,and most preferably, at least 50%) of this inhibitory activity ascompared to that of the murine Pellino-1-FLAG “d133-156-FLAG” mutant asmeasured in the NF-kB-dependent IL-8 promoter-luciferase reporter geneassays of Example 2.

[0067] The term “Pellino polypeptide activity,” as used herein, includesany one or more of the following: stimulation of MAP kinase-activatedsignaling pathways, protease-substrate activity, and host defensiveactivity against pathogens, as well as the ex vivo and in vivoactivities of wild type Pellino polypeptides. The term “Pellinopolypeptide dominant-negative activity,” as used herein, includesinhibition of MAP kinase-activated signaling pathways, anti-inflammatoryactivity, and the ability to sequester binding partners in the insolublecell fraction, as well as the ex vivo and in vivo activities of mutantPellino polypeptides that demonstrate such inhibitory activities inreporter gene assays. The degree to which individual members of thePellino polypeptide family and fragments and other derivatives of thesepolypeptides exhibit these activities can be determined by standardassay methods, particularly assays such as those described in Examples2, 3, and 4 below. Exemplary assays are disclosed herein; those of skillin the art will appreciate that other, similar types of assays can beused to measure Pellino polypeptide biological activities.

[0068] Another aspect of the biological activity of Pellino polypeptidesis their ability to interact with particular intracellular signalingpathway molecules such as TRAF6, IRAK, and IRAK4, with theRING-finger-like domain of Pellino polypeptides likely involved inbinding to such binding partners. The conserved central domain ofPellino polypeptides interacts with a chymotrypsin-like serine proteasethat cleaves Pellino polypeptides, and the N-terminal portion of Pellinopolypeptides is believed to bind a factor involved in localizing Pellinopeptides in, or transporting them to, the portion of the cellularenvironment that becomes the soluble fraction upon cell lysis. Thus,when the N-terminal portion of Pellino-1 is deleted, this Pellinopolypeptide becomes constitutively localized in the insoluble fraction,but is still be able to inhibit MAP kinase-activated signaling pathways,likely by binding signaling pathway polypeptides via itsRING-finger-like domain. The term “binding partner,” as used herein,includes ligands, receptors, substrates, antibodies, other Pellinopolypeptides, the same Pellino polypeptide (in the case of homotypicinteractions), and any other molecule that interacts with a Pellinopolypeptide through contact or proximity between particular portions ofthe binding partner and the Pellino polypeptide. Because theRING-finger-like domain of Pellino polypeptides is believed to bind to asignaling pathway binding partner, the RING-finger-like domain whenexpressed as a separate fragment from the rest of a Pellino polypeptide,but with enough of the N-terminal domain to allow the Pellinopolypeptide to localize to the insoluble fraction, is expected todisrupt the binding of wild-type Pellino polypeptides to their bindingpartners. Particularly suitable assays to detect or measure the bindingbetween Pellino polypeptides and their binding partners include thebioluminescence resonance energy transfer (BRET), which uses abioluminescent luciferase that is genetically fused to one candidateprotein, such as a Pellino polypeptide, and a green fluorescent proteinmutant fused to another protein of interest, such as IRAK, IRAK4, TRAF6,or other potential binding partners. Interactions between the two fusionproteins can bring the luciferase and green fluorescent protein closeenough for resonance energy transfer to occur, thus changing the colorof the bioluminescent emission. Most preferably, the partner-bindingactivities of Pellino polypeptides can be determined usingprotein-fragment complementation assays, as described in Remy andMichnick, 1999, Proc Natl Acad Sci USA 96: 5394-5399 and in WO 01/00866.

[0069] Pellino polypeptides such as murine and human Pellino-1 and -2and human Pellino-3 polypeptides with the ability to stimulate MAPkinase-activated pathways are believed to play a role in protection ofthe host against viral, bacterial, fungal, and other types of pathogens(innate immune responses). In addition, Pellino polypeptides areinvolved in immune and/or inflammatory diseases or conditions, thatshare as a common feature stimulation of MAP kinase-activated pathwaysand NF-kB- and/or p38-dependent transcription in their etiology. Thetherapeutic effect of stimulation of MAP kinase-activated pathways, forexample by administration of a Pellino polypeptide with wild-typeactivity, or fragments or fusion polypeptides with wild-type activity,or agonists thereof, is shown by the following examples of conditions inwhich the stimulation of NF-kB-dependent transcription is beneficial(Yamamoto and Gaynor, 2001, J Clin Invest 107: 135-142). The NF-kBpathway modulates B-lymphocyte survival, mitogen-dependent cellproliferation, and isotype switching, which lead to the differentiationof B lymphocytes into plasma cells. In addition, NF-kB regulates IL-2production, which increases the proliferation and differentiation of Tlymphocytes, and increases the development of Th1-type helper T cells,promoting cell-mediated immunity. Thus, activation of NF-kB leads to theinduction of multiple genes that regulate the immune response. The NF-kBpathway is also a key mediator of genes involved in the control of thecellular proliferation and apoptosis. Antiapoptotic genes that aredirectly activated by NF-kB include the cellular inhibitors of apoptosis(c-IAP1, c-IAP2, and IXAP), the TNF receptor-associated factors (TRAF1and TRAF2), the Bcl-2 homologue A1/Bfl-1, and IEX-IL. Theseantiapoptotic proteins block the activation of caspase-8, an initiatorprotease, involved at an early step in stimulating the apoptoticpathway, and induction of A1/Bfl-1 expression by NF-kB preventscytochrome c release from mitochondria and activation of caspase-3. Byincreasing the expression of antiapoptotic cellular proteins, NF-kBactivation can thus reduce apoptosis in response to treatment withdifferent chemotherapeutic agents. In addition, NF-kB is involved inprotecting cells from undergoing apoptosis in response to DNA damage orcytokine treatment.

[0070] The therapeutic effect of inhibition of MAP kinase-activatedpathways, for example by administration of a Pellino polypeptide with“dominant-negative” inhibitory activity, or fragments or fusionpolypeptides thereof having “dominant-negative” inhibitory activity, orother antagonists of Pellino polypeptides having wild-type activity, isshown by the following examples of conditions in which the inhibition ofNF-kB-dependent transcription is beneficial (Yamamoto and Gaynor, 2001,J Clin Invest 107: 135-142). NF-kB regulates host inflammatory responsesby increasing the expression of specific cellular genes, including genesencoding at least 27 different cytokines and chemokines. Cytokines thatare stimulated by NF-kB, such as IL-1beta and TNF-alpha, can alsodirectly activate the NF-kB pathway, thus establishing a positiveautoregulatory loop that can amplify the inflammatory response andincrease the duration of chronic inflammation. NF-kB also stimulates theexpression of enzymes whose products contribute to the pathogenesis ofthe inflammatory process, including the inducible form of nitric oxidesynthase (iNOS), which generates nitric oxide (NO), and the induciblecyclooxygenase (COX-2), which generates prostanoids. Activation of theNF-kB pathway is involved in the pathogenesis of chronic inflammatorydiseases, such as asthma, rheumatoid arthritis, and inflammatory boweldisease, and other diseases in which inflammation plays a role, such asatherosclerosis and Alzheimer's disease. Several lines of evidencesuggest that NF-kB activation of cytokine genes is an importantcontributor to the pathogenesis of asthma, which is characterized by theinfiltration of inflammatory cells and the dysregulation of manycytokines and chemokines in the lung. Cytokines, such as TNF-alpha, thatactivate NF-kB are elevated in the synovial fluid of patients withrheumatoid arthritis and contribute to the chronic inflammatory changesand synovial hyperplasia seen in the joints of these patients. Increasesin the production of proinflammatory cytokines by both lymphocytes andmacrophages has also been implicated in the pathogenesis of inflammatorybowel diseases, including Crohn's disease and ulcerative colitis. NF-kBactivation is seen in mucosal biopsy specimens from patients with activeCrohn's disease and ulcerative colitis. Treatment of patients withinflammatory bowel diseases with steroids decreases NF-kB activity inbiopsy specimens and reduces clinical symptoms. These results suggestthat stimulation of the NF-kB pathway may be involved in the enhancedinflammatory response associated with these diseases. Atherosclerosis istriggered by numerous insults to the endothelium and smooth muscle ofthe damaged vessel wall. A large number of growth factors, cytokines,and chemokines released from endothelial cells, smooth muscle,macrophages, and lymphocytes are involved in this chronic inflammatoryand fibroproliferative process. Regulation of genes involved in theinflammatory response and in the control of cellular proliferation byNF-kB likely plays an important role in the initiation and progressionof atherosclerosis. Abnormalities in the regulation of the NF-kB pathwaymay be involved in the pathogenesis of Alzheimer's disease. For example,NF-kB immunoreactivity is found predominantly in and around earlyneuritic plaque types in Alzheimer's disease, whereas mature plaquetypes show vastly reduced NF-kB activity. Thus, NF-kB activation may beinvolved in the initiation of neuritic plaques and neuronal apoptosisduring the early phases of Alzheimer's disease. Other conditions inwhich inflammation plays a role and which are expected to be amelioratedby decreases in MAP kinase-activated pro-inflammatory signaling pathwaysinclude osteoporosis, stroke, multiple sclerosis, and multiple myeloma.Additional examples of diseases involving inflammation and/orinflammatory cellular responses are described U.S. Pat. No. 6,204,261 atcolumn 206, line 25, through column 207, line 44; this material fromU.S. Pat. No. 6,204,261 is incorporated by reference herein. In additionto a role in the pathogenesis of diseases characterized by increases inthe host inflammatory response, constitutive activation of the NF-kBpathway has also been implicated in the pathogenesis of some humancancers. Abnormalities in the regulation of the NF-kB pathway arefrequently seen in a variety of human malignancies including leukemias,lymphomas, and solid tumors. These abnormalities result inconstitutively high levels of NF-kB in the nucleus of a variety oftumors including breast, ovarian, prostate, and colon cancers. Themajority of these changes are likely due to alterations in regulatoryproteins that activate signaling pathways that lead to activation of theNF-kB pathway. Preventing, blocking, and/or inhibiting the interactionsbetween Pellino polypeptides and their binding partners is an aspect ofthe invention and provides methods for treating or ameliorating thesediseases and conditions through the use of inhibitors of wild-typePellino activities such as stimulation of NF-kB-dependent transcription.

[0071] Polynucleotide Molecules

[0072] In a particular embodiment, the invention relates to certainisolated polynucleotide molecules that are free from contaminatingendogenous material. “Polynucleotide molecule” refers to polynucleotidemolecules in the form of separate fragments or as a component of largerpolynucleotide constructs. The polynucleotide molecules have preferablybeen derived from DNA or RNA isolated at least once in substantiallypure form and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical-methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

[0073] Polynucleotide molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. Genomic DNA may be isolated by conventional techniques, e.g.,using the cDNA of SEQ ID NOs:1, 3, 5, or 7, or a suitable fragmentthereof, as a probe. The DNA molecules of the invention include DNAsencoding full length Pellino polypeptides as well as polynucleotides andfragments thereof. The polynucleotides of the invention arepreferentially derived from human sources, but the invention includesthose derived from non-human species, as well.

[0074] The present invention encompasses murine Pellino-1 DNA having thepolynucleotide sequence of SEQ ID NO:1 and the polypeptide encoded bythe DNA of SEQ ID NO:1 having the amino acid sequence of SEQ ID NO:2.The polypeptide having amino acids 132 through 189 of SEQ ID NO:2 is apotential target site for protease action for a member of thechymotrypsin family of proteases. The present invention furtherencompasses human Pellino-1 DNA having the polynucleotide sequence ofSEQ ID NO:3 and the polypeptide encoded by the DNA of SEQ ID NO:3 havingthe amino acid sequence of SEQ ID NO:4. A potential protease target siteis also found in this polypeptide, corresponding to amino acids 132through 189 of SEQ ID NO:4. Further encompassed by the present inventionis the DNA of murine Pellino-2 and having the polynucleotide sequence ofSEQ ID NO:5, and polypeptide encoded by SEQ ID NO:5 shown in SEQ IDNO:6. The potential protease target site is located between amino acids133 and 190 of murine Pellino-2. Similarly, the potential proteasetarget site of human Pellino-2 is likely to be between amino acids 134and 191 of SEQ ID NO:8. The above described protease target sequences ofSEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 may vary by one or more aminoacids. Thus, the amino terminus of the protease target region for SEQ IDNO:2 and SEQ ID NO:4 may occur from amino acid 130 through 134 and thecarboxy terminus of the target region from amino acid 187 through 191.Similarly, for SEQ ID NO:6, the amino terminus of the protease targetregion occurs from amino acid 131 through amino acid 135 (amino acids132 through 136 of SEQ ID NO:8) and the carboxy terminus from amino acid188 through 192 (amino acids 189 through 193 of SEQ ID NO:8).

[0075] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA can vary from thatshown in SEQ ID NO:1, and still encode a polypeptide having the aminoacid sequence of SEQ ID NO:2. Such variant DNAs can result from silentmutations that occur naturally, or during PCR amplification, or they canbe the product of deliberate mutagenesis of a native sequence. The sameis true for the DNAs depicted in SEQ ID NOs: 3, 5 and 7.

[0076] The invention thus provides isolated DNAs encoding polypeptidesof the invention, selected from: (a) a DNA comprising the nucleotidesequence of SEQ ID NO:1; (b) a DNA comprising the nucleotide sequence ofSEQ ID NO:3; (c) a DNA comprising the nucleotide sequence of SEQ IDNO:5; (d) a DNA encoding the polypeptides encoded by the DNA of (a), (b)or (c); (e) a DNA capable of hybridization to the DNA of (a), (b) or (c)under conditions of moderate stringency and which encodes a polypeptideof the invention; (f) a DNA capable of hybridization to the DNA of (a),(b), or (c) under conditions of high stringency and which encodes apolypeptide of the invention, and (g) a DNA which is degenerate as aresult of the genetic code to a DNA defined in (a), (b), (c), (d), (e),or (f) and which encodes a polypeptide of the invention.

[0077] The basic parameters affecting the choice of hybridizationconditions and guidance for devising suitable conditions are set forthby Sambrook et al., 1989. As used herein, conditions of moderatestringency can be readily determined by those having ordinary skill inthe art based on, for example, the length and/or base composition of theDNA. For hybridizing probes longer than about 100 nucleotides withfilter-bound target DNA or RNA, one way of achieving moderatelystringent conditions involves the use of a prewashing solutioncontaining 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization bufferof about 50% formamide, 6×SSC, and a hybridization temperature of about42° C. (or other similar hybridization solutions, such as one containingabout 50% formamide, with a hybridization temperature of about 42° C.),and washing conditions of about 60° C., in 0.5×SSC, 0.1% SDS. Conditionsof high stringency can also be readily determined by the skilled artisanbased on, for example, the length and base composition of the DNA.Generally, such conditions are defined as hybridization conditions asabove, but with washing at approximately 68° C., 0.2×SSC, 0.1% SDS. Itshould be understood that the wash temperature and wash saltconcentration can be adjusted as necessary to achieve a desired degreeof stringency by applying the basic principles that govern hybridizationreactions and duplex stability, as known to those skilled in the art(see, e.g., Sambrook et al., 1989). It should be further understood thathybridization conditions for oligonucleotide probes of defined lengthand sequence can be designed by applying formulae known in the art(e.g., see Sambrook et al., 1989, at 11.4511.47).

[0078] Also included as an embodiment of the invention is DNA encodingpolypeptide fragments that have at least one activity of Pellinopolypeptides, and DNA encoding polypeptides of at least about 16 aminoacids, or of at least about 32 amino acids, which polypeptides areuseful as immunogens. DNAs encoding polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), are also included, as describedbelow. For example, the IL-1R-homologous domain may be useful as adominant negative regulator of IL-1R signaling, or in an assay toidentify small molecules that can inhibit or otherwise regulate IL-1signaling.

[0079] In another embodiment, the DNA molecules of the invention alsocomprise polynucleotides that are at least 80% identical to a nativesequence, and polynucleotide molecules that are at least 85% identicalto a native molecule. Also contemplated are embodiments in which a DNAmolecule is at least 90% identical, at least 95% identical, at least 98%identical, at least 99% identical, or at least 99.9% identical to anative sequence. Percent identity is defined as the number of alignedsymbols, i.e. nucleotides or amino acids, which are identical, dividedby the total number of symbols in the shorter of the two sequences. Thedegree of homology (percent identity) between two sequences may bedetermined by using the alignment method of Needleman and Wunsch (J.Mol. Biol. 48:443, 1970) as revised by Smith and Waterman (Adv. Appl.Math 2:482, 1981), with a unary comparison matrix (containing a value of1 for identities and 0 for nonidentities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess (Nucl. Acids. Res.14:6745, 1986) as described by Schwartz and Dayhoff (Atlas of ProteinSequence and Structure, National Biomedical Research Foundation, pp.353-358, 1979) for amino acids. Preferably, the comparison is done usinga computer program. An exemplary, preferred computer program is theGenetics Computer Group (GCG; Madison, Wis.) Wisconsin package version10.0 program, ‘GAP.’ The preferred default parameters for the ‘GAP’program includes: (1) The GCG implementation of the previously statedcomparison matrixes for nucleotides and amino acids; (2) a penalty of 30for each gap and an additional penalty of 1 for each symbol in each gapfor amino acid sequences, or penalty of 50 for each gap and anadditional penalty of 3 for each symbol in each gap for nucleotidesequences; (3) no penalty for end gaps; and (4) no maximum penalty forlong gaps. Other programs used by one skilled in the art of sequencecomparison may also be used.

[0080] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide. Inaddition, DNAs that encode various additions or substitutions of aminoacid residues or sequences, or deletions of terminal or internalresidues or sequences are encompassed by the invention.

[0081] Examples of such DNAs include those that have been modified tofacilitate expression of a polypeptide with an altered N-linkedglycosylation site or KEX-2 protease site, as well as those in whichcodons that encode Cys residues that are not necessary for biologicalactivity are eliminated or altered to encode another amino acid. Theseand other variant peptides are disclosed herein; DNAs encoding them arealso encompassed by the invention.

[0082] The invention also provides isolated DNAs useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding a Pellinopolypeptide, or desired fragment thereof may be subcloned into anexpression vector for production of the polypeptide or fragment. The DNAsequence advantageously is fused to a sequence encoding a suitableleader or signal peptide.

[0083] The desired DNA fragment may be chemically synthesized usingknown techniques. DNA fragments also may be produced by restrictionendonuclease digestion of a full length cloned DNA sequence, andisolated by electrophoresis on agarose gels. If necessary,oligonucleotides that reconstruct the 5′ or 3′ terminus to a desiredpoint may be ligated to a DNA fragment generated by restriction enzymedigestion. Such oligonucleotides may additionally contain a restrictionendonuclease cleavage site upstream of the desired coding sequence, andposition an initiation codon (ATG) at the N-terminus of the codingsequence.

[0084] The well-known polymerase chain reaction (PCR) procedure also maybe employed to isolate and amplify a DNA encoding a desired protein orfragment thereof. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

[0085] The present invention also provides genes corresponding to thenucleic acid sequences disclosed herein. “Corresponding genes” or“corresponding genomic nucleic acids” are the regions of the genome thatare transcribed to produce the mRNAs from which cDNA nucleic acidsequences are derived and can include contiguous regions of the genomenecessary for the regulated expression of such genes. Correspondinggenes can therefore include but are not limited to coding sequences, 5′and 3′ untranslated regions, alternatively spliced exons, introns,promoters, enhancers, and silencer or suppressor elements. Correspondinggenomic nucleic acids can include 10000 basepairs (more preferably, 5000basepairs, still more preferably, 2500 basepairs, and most preferably,1000 basepairs) of genomic nucleic acid sequence upstream of the firstnucleotide of the genomic sequence corresponding to the initiation codonof the Pellino polypeptide coding sequence, and 10000 basepairs (morepreferably, 5000 basepairs, still more preferably, 2500 basepairs, andmost preferably, 1000 basepairs) of genomic nucleic acid sequencedownstream of the last nucleotide of the genomic sequence correspondingto the termination codon of the Pellino polypeptide coding sequence. Thecorresponding genes or genomic nucleic acids can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials. An “isolated gene” or “an isolated genomic nucleicacid” is a genomic nucleic acid that has been separated from theadjacent genomic sequences present in the genome of the organism fromwhich the genomic nucleic acid was isolated.

[0086] Polypeptides and Fragments Thereof

[0087] The invention encompasses polypeptides and fragments thereof invarious forms, including those that are naturally occurring or producedthrough various techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

[0088] The polypeptides of the invention include full length proteinsencoded by the nucleic acid sequences set forth above. Full lengthpolypeptides comprise an amino acid sequence as depicted in SEQ ID NOs:2, 4, and 6, with useful fragments comprising amino acids 132 to 289 ofSEQ ID NOs:2 and 4, and amino acids 133 to 190 of SEQ ID NO:6. Asmentioned above, the N-terminal and C-terminal amino acids of these andother fragments can vary about two amino acids from those given (i.e.,the N-terminus can vary from amino acids 130 to 134 of SEQ ID NOs:2 and4 and 131 to 135 of SEQ ID NO:6; and the C-terminus can vary from aminoacids 187 to 191 of SEQ ID NOs:2 and 4 and 188 to 192 of SEQ ID NO:6).

[0089] The inventive peptides and fragments thereof may be recombinantlyexpressed as an intracellular polypeptide, preferably in non-mammaliancells. Such peptides may be obtained by isolating cells that express thepolypeptide from the culture medium (e.g., by centrifugation orfiltration), solubilizing the cells, and isolating the peptide from thesolubilized cells. Choice of solubilization techniques will depend onthe cells used for expression. Purification of the polypeptide fromrecombinant host cells is facilitated by expression of the polypeptideas a fusion protein with a tag protein as discussed herein.

[0090] The inventive peptides and fragments thereof may also berecombinantly expressed as a soluble polypeptide capable of beingsecreted from the cells in which it is made. Such soluble peptides maybe obtained by separating intact cells that express the solublepolypeptide from the culture medium (e.g., by centrifugation orfiltration), and isolating the soluble peptide from the medium(supernatant). Purification of the polypeptides from recombinant hostcells is facilitated by expression of the polypeptide as a secretedprotein, which can be useful in obtaining large amounts of the solublepolypeptide as a therapeutic or diagnostic agent, or for use in assays.Because the N-terminus and C-terminus of recombinantly expressedpolypeptides may vary by several amino acids, including from about 1amino acid to about 10 amino acids, the polypeptides of this inventioncan vary accordingly.

[0091] The inventive polypeptides thus include, but are not limited to:(a) polypeptides comprising amino acids ×1 to ×2, wherein ×1 is any ofthe amino acids in positions 1 through 10 of SEQ ID NO:2, and ×2 is anyof the amino acids in positions 408 through 418 of SEQ ID NO:2; (b)polypeptides comprising amino acids ×1 to ×2, wherein ×1 is any of theamino acids in positions 1 through 10 of SEQ ID NO:4, and ×2 is any ofthe amino acids in positions 408 through 418 of SEQ ID NO:4; and (c)polypeptides comprising amino acids ×1 to ×2, wherein ×1 is any of theamino acids in positions 1 through 10 of SEQ ID NO:6, and ×2 is any ofthe amino acids in positions 409 through 419 of SEQ ID NO:6.Polypeptides similar to any of the foregoing may also be derived fromSEQ ID NO:8.

[0092] Other embodiments include polypeptides comprising: (a) aminoacids ×1 to ×2, wherein ×1 is any of the amino acids in positions 1through 10 of SEQ ID NO:2, and ×2 is any of the amino acids in positions187 through 191 of SEQ ID NO:2; (b) amino acids ×1 to ×2, wherein ×1 isany of the amino acids in positions 1 through 10 of SEQ ID NO:4, and ×2is any of the amino acids in positions 187 through 191 of SEQ ID NO:4;and (c) amino acids ×1 to ×2, wherein ×1 is any of the amino acids inpositions 1 through 10 of SEQ ID NO:6, and ×2 is any of the amino acidsin positions 188 through 192 of SEQ ID NO:6.

[0093] The invention also comprehends polypeptides comprising: (a) aminoacids ×1 to ×2, wherein ×1 is any of the amino acids in positions 1through 10 of SEQ ID NO:2, and ×2 is any of the amino acids in positions130 through 134 of SEQ ID NO:2; (b) amino acids ×1 to ×2, wherein ×1 isany of the amino acids in positions 1 through 10 of SEQ ID NO:4, and ×2is any of the amino acids in positions 130 through 134 of SEQ ID NO:4;and (c) amino acids ×1 to ×2, wherein ×1 is any of the amino acids inpositions 1 through 10 of SEQ ID NO:6, and ×2 is any of the amino acidsin positions 129 through 133 of SEQ ID NO:6.

[0094] Additional embodiments include polypeptides comprising: (a) aminoacids ×1 to ×2, wherein ×1 is any of the amino acids in positions 130through 134 of SEQ ID NO:2, and ×2 is any of the amino acids inpositions 408 through 418 of SEQ ID NO:2; (b) amino acids ×1 to ×2,wherein ×1 is any of the amino acids in positions 130 through 134 of SEQID NO:4, and ×2 is any of the amino acids in positions 408 through 418of SEQ ID NO:4; and (c ) amino acids ×1 to ×2, wherein ×1 is any of theamino acids in positions 129 through 133 of SEQ ID NO:6, and ×2 is anyof the amino acids in positions 409 through 419 of SEQ ID NO:6.

[0095] Also included within the scope of the invention are polypeptidescomprising: (a) amino acids ×1 to ×2, wherein ×1 is any of the aminoacids in positions 187 through 191 of SEQ ID NO:2, and ×2 is any of theamino acids in positions 408 through 418 of SEQ ID NO:2; (b) amino acids×1 to ×2, wherein ×1 is any of the amino acids in positions 187 through191 of SEQ ID NO:4, and ×2 is any of the amino acids in positions 408through 418 of SEQ ID NO:4; and (c) amino acids ×1 to ×2, wherein ×1 isany of the amino acids in positions 188 through 192 of SEQ ID NO:6, and×2 is any of the amino acids in positions 409 through 419 of SEQ IDNO:6. Polypeptides similar to any of the foregoing may also be derivedfrom SEQ ID NO:8.

[0096] The invention also provides Pellino polypeptides and fragmentsthereof that retain a desired activity. Particular embodiments aredirected to polypeptide fragments that retain the ability to bind amember of the chymotrypsin family of proteases. Such a fragment may be asoluble polypeptide, as described above. In another embodiment, thepolypeptides and fragments advantageously include regions that areconserved in the Pellino family as described above.

[0097] Also provided herein are polypeptide fragments comprising atleast 8, 12, 16, or at least 32, contiguous amino acids of the sequenceof SEQ ID NO:2. Such polypeptide fragments may be employed as immunogensin generating antibodies, as small molecule agonists or antagonists ofPellino activity, and in various assays for Pellino activity.

[0098] Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein. Variants may exhibitamino acid sequences that are at least 80% identical, or at least about85% identical, to the native polypeptide disclosed herein. Alsocontemplated are embodiments in which a polypeptide or fragmentcomprises an amino acid sequence that is at least 90% identical, atleast 95% identical, at least 98% identical, at least 99% identical, orat least 99.9% identical to the preferred polypeptide or fragmentthereof. Percent identity may be determined as described previouslyherein.

[0099] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring, shortenedform of the protein. As mentioned above, variations attributable toproteolysis include, for example, differences in the N- or C-terminiupon expression in different types of host cells, due to proteolyticremoval of one or more terminal amino acids from the protein (generallyfrom about one to about five terminal amino acids) or other differencesin protein expression. Proteins in which differences in amino acidsequence are attributable to genetic polymorphism (allelic variationamong individuals producing the protein) are also contemplated herein.

[0100] Other variants include fusion proteins, such as those prepared byexpression in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins include fusion proteins that will formoligomers, such as a Pellino/Fc fusion protein (for example, asdescribed in U.S. Pat. No. 5,962,406, issued Oct. 5, 1999), or a zipperfusion protein (U.S. Pat. No. 5,716,805, issued Feb. 10, 1998). Further,fusion proteins can comprise peptides added to facilitate purificationand identification (often referred to as tag proteins). Such peptidesinclude, for example, poly-His or the antigenic identification peptidesdescribed in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology6:1204, 1988. Additional, useful tag proteins include green fluorescentprotein (GFP; Chalfie et al., Science 263:802, 1994), an N-terminalpeptide that contains recognition sites for a monoclonal antibody, aspecific endopeptidase, and a site-specific protein kinase (PKA; Blanarand Rutter, Science 256:1014, 1992), birA (Altman et al., Science274:94, 1996) and glutathione S transferase (GST: Smith and Johnson,Gene 67:31, 1988).

[0101] One such tag peptide is the FLAG peptide, which is highlyantigenic and provides an epitope reversibly bound by a specificmonoclonal antibody, enabling rapid assay and facile purification ofexpressed recombinant protein. A murine hybridoma designated 4E11produces a monoclonal antibody that binds the FLAG peptide in thepresence of certain divalent metal cations, as described in U.S. Pat.No. 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cellline has been deposited with the American Type Culture Collection underaccession no. HB 9259. Monoclonal antibodies that bind the FLAG peptideare available from Eastman Kodak Co., Scientific Imaging SystemsDivision, New Haven, Conn.

[0102] Another useful tag peptide is the GST peptide, which bindsglutathione, also facilitating purification of expressed recombinantprotein. Recombinant protein can be purified by affinity chromatographyusing a suitable chromatography matrix to which has been attachedglutathione, as described in Smith and Johnson, supra, herebyincorporated by reference. Suitable chromatography matrixes includeGlutathione-Agarose beads (Pharmacia). Recombinant protein can be elutedwith an excess of glutathione. Alternatively, a specific enzymaticcleavage site (such as a thrombin cleavage site) can be included n therecombinant fusion protein, and the desired polypeptide removed from theaffinity matrix by treatment with the enzyme that cleaves the fusionprotein at the cleavage site.

[0103] Among the variant polypeptides provided herein are variants ofnative polypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds abinding partner with essentially the same binding affinity as does thenative form. Binding affinity can be measured by conventionalprocedures, e.g., as described in U.S. Pat. No. 5,512,457 and as setforth below. Variants include polypeptides that are substantiallyhomologous to the native form, but which have an amino acid sequencedifferent from that of the native form because of one or more deletions,insertions or substitutions. Particular embodiments include, but are notlimited to, polypeptides that comprise from one to ten deletions,insertions or substitutions of amino acid residues, when compared to anative sequence. A given amino acid may be replaced, for example, by aresidue having similar physiochemical characteristics. Examples of suchphysiochemically conservative substitutions include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother; substitutions of one polar residue for another, such as betweenLys and Arg, Glu and Asp, or Gln and Asn; or substitutions of onearomatic residue for another, such as Phe, Trp, or Tyr for one another.Other substitutions, e.g., involving substitutions of entire regionshaving similar hydrophobicity characteristics, are well known.

[0104] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., CHO or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Further, a given preparation mayinclude multiple differentially glycosylated species of the protein.Expression of polypeptides of the invention in bacterial expressionsystems, such as E. coli, provides non-glycosylated molecules. Glycosylgroups can also be removed through conventional chemical or enzymaticmethods, in particular those utilizing glycopeptidase. In general,glycosylated polypeptides of the invention can be incubated with a molarexcess of glycopeptidase (Boehringer Mannheim). Recombinant technologycan also be applied to reduce glycosylation that occurs in eukaryoticexpression systems, for example, as described in U.S. Pat. No. 5,071,972and EP 276,846, hereby incorporated by reference. Other variants areprepared by modification of adjacent dibasic amino acid residues, toenhance expression in yeast systems in which KEX2 protease activity ispresent, as disclosed in EP 212,914. In another example of variants,sequences encoding Cys residues that are not essential for biologicalactivity can be altered to cause the Cys residues to be deleted orreplaced with other amino acids, as disclosed in U.S. Pat. No.5,962,406, issued Oct. 5, 1999.

[0105] Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

[0106] Production of Polypeptides and Fragments Thereof

[0107] The present invention also provides recombinant cloning andexpression vectors containing DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising DNA may be used toprepare the polypeptides or fragments of the invention encoded by theDNA. A method for producing polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that procedures forproducing and purifying the expressed polypeptides will vary accordingto such factors as the type of host cells employed, and whether thepolypeptide is membrane-bound or a soluble polypeptide that is secretedfrom the host cell.

[0108] Any suitable expression system may be employed. The vectorsinclude a DNA encoding a polypeptide or fragment of the invention,operably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. An origin of replication thatconfers the ability to replicate in the desired host cells, and aselection gene by which transformants are identified, are generallyincorporated into the expression vector. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence.

[0109] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

[0110] The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide.Accordingly, a protein preparation may include a mixture of proteinmolecules having different N-terminal amino acids, resulting fromcleavage of the signal peptide at more than one site.

[0111] Suitable host cells for expression of polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0112] Mammalian or insect host cell culture systems also may beemployed to express recombinant polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

[0113] A commonly used cell line is dihydrofolate reductase (DHFR)-CHOcells which are auxotrophic for glycine, thymidine and hypoxanthine, andcan be transformed to the DHFR+ phenotype using DHFR cDNA as anamplifiable dominant marker. One such DHFR-CHO cell line, DXB 11, wasdescribed by Urlaub and Chasin (Proc. Natl. Acad. Sci. USA 77:4216,1980). Another exemplary DHFR-CHO cell line is DG44 (see, for example,Kaufman, R. J., Meth. Enzymology 185:537 (1988). Other cell linesdeveloped for specific selection or amplification schemes will also beuseful with the invention.

[0114] Several transfection protocols are known in the art, and arereviewed in Kaufman, R. J., supra. The transfection protocol chosen willdepend on the host cell type and the nature of the gene of interest, andcan be chosen based upon routine experimentation. The basic requirementsof any such protocol are first to introduce DNA encoding the protein ofinterest into a suitable host cell, and then to identify and isolatehost cells which have incorporated the heterologous DNA in a stable,expressible manner. Other useful transfection protocols are discussed inU.S. Pat. No. 6,027,915, issued Feb. 22, 2000. Transfection of cellswith heterologous DNA and selection for cells that have taken up theheterologous DNA and express the selectable marker results in a pool oftransfected cells. Individual cells in these pools will vary in theamount of DNA incorporated and in the chromosomal location of thetransfected DNA. To generate stable cell lines, individual cells can beisolated from the pools and cultured (a process referred to as cloning).

[0115] A method of amplifying the gene of interest is also desirable forexpression of the recombinant protein, and typically involves the use ofa selection marker (reviewed in Kaufman, R. J., supra). Resistance tocytotoxic drugs is the characteristic most frequently used as aselection marker, and can be the result of either a dominant trait(i.e., can be used independent of host cell type) or a recessive trait(i.e., useful in particular host cell types that are deficient inwhatever activity is being selected for). Several amplifiable markersare suitable for use in the inventive expression vectors (for example,as described in Maniatis, Molecular Biology: A Laboratory Manual, ColdSpring Harbor Laboratory, NY, 1989; pgs 16.9-16.14).

[0116] Useful selectable markers for gene amplification indrug-resistant mammalian cells are shown in Table 1 of Kaufman, R. J.,supra (1988), and include DHFR-MTX resistance, P-glycoprotein andmultiple drug resistance (MDR)-various lipophilic cytoxic agents (i.e.,adriamycin, colchicine, vincristine), and adenosine deaminase(ADA)-Xyl-A or adenosine and 2′-deoxycoformycin. Other dominantselectable markers are discussed in U.S. Pat. No. 6,027,915, issued Feb.22, 2000).

[0117] Useful regulatory elements, described previously, can also beincluded in the plasmids or expression vectors used to transfectmammalian cells. The transfection protocol chosen, and the elementsselected for use therein, will depend on the type of host cell used.Those of skill in the art are aware of numerous different protocols andhost cells, and can select an appropriate system for expression of adesired protein, based on the requirements of their selected cellculture system(s).

[0118] A useful high expression vector, pCAVNOT, has been described byMosley et al., Cell 59:335-348, 1989. Other expression vectors for usein mammalian host cells can be constructed as disclosed by Okayama andBerg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSVN1/N4, described by Cosman et al., Nature 312:768, 1984, has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in WO 91/18982, incorporated byreference herein. In yet another alternative, the vectors can be derivedfrom retroviruses.

[0119] Additional useful expression vectors, pFLAG and pDC311, can alsobe used. FLAG technology is centered on the fusion of a low molecularweight (1 kD), hydrophilic, FLAG marker peptide to the N-terminus of arecombinant protein expressed by pFLAG expression vectors. pDC311 isanother specialized vector used for expressing proteins in CHO cells.pDC311 is characterized by a bicistronic sequence containing the gene ofinterest and a dihydrofolate reductase (DHFR) gene with an internalribosome binding site for DHFR translation, an expression augmentingsequence element (EASE), the human CMV promoter, a tripartite leadersequence, and a polyadenylation site.

[0120] A signal peptide may be employed to facilitate secretion of theprotein, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312: 768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

[0121] Puuification

[0122] The “isolated” polypeptides or fragments thereof encompassed bythis invention are polypeptides or fragments that are not in anenvironment identical to an environment in which it or they can be foundin nature. The “purified” polypeptides or fragments thereof encompassedby this invention are essentially free of association with othercellular components, such as unrelated proteins or polypeptides, lipidsand DNA or RNA, for example, as a purification product of recombinantexpression systems such as those described above or as a purifiedproduct from a non-recombinant source such as naturally occurring cellsand/or tissues.

[0123] In one embodiment, the purification of recombinant polypeptidesor fragments can be accomplished by expressing the inventivepolypeptide(s) as a fusion protein with a peptide (often referred to asa tag peptide) for which an affinity purification scheme is known in theart. Such fusion partners can include the poly-His or other tag peptidesdescribed above as well as an Fc moiety or a zipper moiety.

[0124] With respect to purification, as is known to the skilled artisan,procedures for purifying a recombinant polypeptide or fragment will varyaccording to such factors as the type of host cells employed and whetheror not the recombinant polypeptide or fragment is secreted into theculture medium. In general, the recombinant polypeptide or fragment canbe isolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps.

[0125] As to specific ways to accomplish these steps, the culture mediumfirst can be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. Following the concentration step, the concentratecan be applied to a purification matrix such as a gel filtration medium.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification.

[0126] Alternatively, a cation exchange step can be employed. Suitablecation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. In addition, a chromatofocusingstep can be employed. Alternatively, a hydrophobic interactionchromatography step can be employed. Suitable matrices can be phenyl oroctyl moieties bound to resins. In addition, affinity chromatographywith a matrix which selectively binds the recombinant protein can beemployed. Examples of such resins employed are lectin columns, dyecolumns, and metal-chelating columns.

[0127] Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel or polymer resin having pendant methyl, octyl,octyldecyl or other aliphatic groups) can be employed to further purifythe polypeptides. Some or all of the foregoing purification steps, invarious combinations, are well known and can be employed to provide anisolated and purified recombinant protein.

[0128] It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. In this aspect of the invention,binding proteins, such as antibodies against Pellino or other moleculesthat bind Pellino can be bound to a solid phase support such as a columnchromatography matrix or a similar substrate suitable for identifying,separating, or purifying Pellino. Adherence of Pellino to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with Pellino binding proteins (orother Pellino-binding molecules) and held in the incubation vesselthrough a magnetic field.

[0129] Solutions containing Pellino polypeptides are contacted with thesolid phase under conditions promoting binding of Pellino polypeptidesto the binding partner; unbound material is then washed away. Methods ofreleasing positively selected peptides from the solid phase are known inthe art and encompass, for example, use of a high salt elution bufferfollowed by dialysis into a lower salt buffer, or by changing pH (orother characteristics depending on the affinity matrix utilized), orcompetitive removal using a naturally occurring substrate of theaffinity moiety. The methods are preferably non-injurious to the Pellinopolypeptides.

[0130] In one exemplary method, solutions containing Pellinopolypeptides of the invention first can be incubated with a biotinylatedPellino binding partner. Incubation periods are typically at least onehour in duration to ensure sufficient binding to polypeptides of theinvention. The resulting mixture then is passed through a column packedwith avidin-coated beads, whereby the high affinity of biotin for avidinprovides the binding of the Pellino polypeptides to the beads. Use ofavidin-coated beads is known in the art. See Berenson, et al. J. Cell.Biochem., 10D:239 (1986). Washing of unbound material and the release ofthe bound cells are performed using conventional methods.

[0131] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0132] Uses of Pellino Nucleic Acid or Oligonucleotides

[0133] Among the uses of nucleic acids of the invention is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a nucleic acid sequence. Inother embodiments, a nucleic acid fragment comprises at least 30, or atleast 60, contiguous nucleotides of a nucleic acid sequence.

[0134] Because homologs of Pellino proteins from other mammalian speciesare contemplated herein, probes based on the DNA sequence of SEQ IDNOs:1, 3, 5, 7, or 11 may be used to screen cDNA libraries derived fromother mammalian species, using conventional cross-species hybridizationtechniques.

[0135] Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

[0136] All or a portion of the nucleic acids of SEQ ID NOs:1, 3, 5, 7,or 11, including oligonucleotides, can be used by those skilled in theart using well-known techniques to identify the human chromosome, andthe specific locus thereof, that contains the DNA of a Pellino familymember. Useful techniques include, but are not limited to, using thesequence or portions, including oligonucleotides, as a probe in variouswell-known techniques such as radiation hybrid mapping (highresolution), in situ hybridization to chromosome spreads (moderateresolution), and Southern blot hybridization to hybrid cell linescontaining individual human chromosomes (low resolution).

[0137] For example, chromosomes can be mapped by radiationhybridization, using primers that lie within a putative exon of the geneof interest and which amplify a product from human genomic DNA, but donot amplify genomic DNA from other species. The results of the PCR areconverted into a data vector that is scored and the chromosomalassignment and placement relative to known Sequence Tag Site (STS)markers on the radiation hybrid map is determined. Human Pellino-1 mapsto chromosome 2, places 7.15 cR from WI-6130.

[0138] The nucleic acid of SEQ ID NOs 1, 3, 5, 7, or 11, or a fragmentthereof can be used by one skilled in the art using well-knowntechniques to analyze abnormalities associated with gene mapping to achromosome that comprises a gene encoding Pellino. This enables one todistinguish conditions in which this marker is rearranged or deleted. Inaddition, nucleotides of SEQ ID NOs:1, 3, 5, 7, or 11, or a fragmentthereof can be used as a positional marker to map other genes of unknownlocation.

[0139] The DNA may be used in developing treatments for any disordermediated (directly or indirectly) by defective, or insufficient amountsof, the genes corresponding to the nucleic acids of the invention.Disclosure herein of native nucleotide sequences permits the detectionof defective genes, and the replacement or supplementation thereof withnormal genes, by various gene therapy techniques that are known in theart. Defective genes may be detected in in vitro diagnostic assays, andby comparison of a native nucleotide sequence disclosed herein with thatof a gene derived from a person suspected of harboring a defect in thisgene.

[0140] Inhibitory nucleic acids that reduce Pellino polypeptide activityare also encompassed within the scope of the invention, such asantisense nucleic acids, ribozymes, and ‘interfering’ or ‘silencing’double-stranded RNA molecules. Examples of inhibitory (antagonistic)nucleic acids include antisense or sense oligonucleotides comprising asingle-stranded nucleic acid sequence capable of binding to target mRNAor DNA sequences. Antisense or sense oligonucleotides, according to thepresent invention, comprise a fragment of DNA of SEQ ID NOs:1, 3, 5, 7,or 11. Such a fragment generally comprises at least about 17nucleotides, preferably from about 17 to about 30 nucleotides. Theability to derive an antisense or a sense oligonucleotide, based upon acDNA sequence encoding a given protein is described in, for example,Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988). Binding of antisense or senseoligonucleotides to target nucleic acid sequences results in theformation of complexes that block or inhibit protein expression by oneof several means, as discussed in U.S. Pat. No. 5,783,665, issued Jul.21, 1998. Organic moieties and other moieties that increases affinity ofthe oligonucleotide for a target nucleic acid sequence, or intercalatingagents, may be attached to sense or antisense oligonucleotides to modifybinding specificities of the antisense or sense oligonucleotide for thetarget nucleotide sequence. The antisense or sense oligonucleotides maybe introduced into a cell containing the target nucleic acid sequence byany gene transfer method, including, for example, lipofection,CaPO4-mediated DNA transfection, electroporation, or by using genetransfer vectors such as Epstein-Barr virus. Sense or antisenseoligonucleotides also may be introduced into a cell containing thetarget nucleotide sequence by formation of a conjugate with a ligandbinding molecule, as described in WO 91/04753. Alternatively, a sense oran antisense oligonucleotide may be introduced into a cell containingthe target nucleic acid sequence by formation of anoligonucleotide-lipid complex, as described in WO 90/10448.

[0141] Ribozyme molecules designed to bind to and catalytically cleavePellino mRNA transcripts can also be used to prevent translation ofPellino mRNA and expression of Pellino polypeptides. (See, e.g., PCTInternational Publication WO90/11364 and U.S. Pat. No. 5,824,519). Theribozymes that can be used in the present invention include hammerheadribozymes (Haseloff and Gerlach, 1988, Nature, 334:585-591), RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA; International Patent Application No. WO 88/04300; Been andCech, 1986, Cell, 47:207-216). As in the antisense approach, theribozymes can be composed of modified oligonucleotides (e.g. forimproved stability, targeting, etc.) and should be delivered to cellswhich express the Pellino polypeptide in vivo. A preferred method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy endogenous Pellino messages and inhibit translation. Becauseribozymes, unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

[0142] The “RNA interference” (“RNAi”) technique (Grishok, Tabara, andMello, 2000, Science 287: 2494-2497), also called “RNA silencing”, canbe used to inhibit the expression of particular target genes such asPellino gene by introducing into cells double-stranded small interferingRNAs (siRNAs) that specifically bind to Pellino nucleic acids (Martinezet al, 2002, Cell 110:563 and Elbashir et al, 2002, Methods 26:199-213). This approach can be adapted for use in humans provided theconstructs encoding siRNAs or precursors thereof are directlyadministered or targeted to the required site in vivo using appropriatevectors such as viral vectors. Double-stranded small interfering RNAs(siRNAs) that bind to Pellino nucleic acids can be formed of RNA strands17 to 30 bases in length, or 19 to 25 bases in length, or 19, 20, 21,22, or 23 bases in length.

[0143] The inventive DNAs will also be useful in the development oftransgenic and/or knockout cells and animals. Those of ordinary skill inthe art are aware of various methods by which such cells or animals canbe prepared; an exemplary method is given in U.S. Pat. No. 5,565,321,issued Oct. 15, 1996. The techniques described therein can be used withthe inventive sequences by the application of routine experimentation.

[0144] Uses of Pellino Polypeptides

[0145] Because Pellino proteins are homologous to the Pellino proteinsof Drosophila, an important molecule in the signaling cascade for theIL-1R/Toll family of receptors, small molecule inhibitors of itsfunction or protein associations(or antisense or other inhibitors of itssynthesis) may be useful in treating autoimmune and/or inflammatorydisorders. Accordingly, the Pellino polypeptides of the presentinvention may be used in a screening assay to identify compounds andsmall molecules which inhibit (antagonize) or enhance (agonize)activation of the polypeptides of the instant invention.

[0146] Thus, for example, polypeptides of the invention may be used toidentify antagonists and agonists from cells, cell-free preparations,chemical libraries, and natural product mixtures. The antagonists andagonists may be natural or modified substrates, ligands, enzymes,receptors, etc. of the polypeptides of the instant invention, or may bestructural or functional mimetics of the polypeptides. Potentialantagonists of the instant invention may include small molecules,peptides and antibodies that bind to and occupy a binding site of theinventive polypeptides or a binding partner thereof, causing them to beunavailable to bind to their natural binding partners and thereforepreventing normal biological activity. Potential agonists include smallmolecules, peptides and antibodies which bind to the instantpolypeptides or binding partners thereof, and elicit the same orenhanced biologic effects as those caused by the binding of thepolypeptides of the instant invention. Peptide agonists and antagonistsof the polypeptides of the invention can be identified and utilizedaccording to known methods (see, for example, WO 00/24782 and WO01/83525, which are incorporated by reference herein).

[0147] Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physicochemical andpharmacological properties which enhance cell penetration, resistdegradation and prolong their physiological half-lives (Gibbs, J.,Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994)).Antibodies, which include intact molecules as well as fragments such asFv, Fab, and F(ab′)2 fragments, single-chain antibodies such as scFv, aswell as recombinant molecules derived therefrom, may be used to bind toand inhibit the polypeptides of the instant invention by blocking thepropagation of a signaling cascade. Nucleic acids encoding single-chainantibodies such as scFv can be introduced into cells in order to expressintracellularly such single-chain antibodies, where the Fv domain isoptionally linked to domains such as Fc, the IgM transmembrane domain,or an endoplasmic reticulum retention signal (Teasdale and Jackson,1996, Annu Rev Cell Dev Biol 12: 27-54). It is preferable that theantibodies are humanized, and more preferable that the antibodies arehuman. The antibodies of the present invention may be prepared by any ofa variety of well-known methods.

[0148] Specific screening methods are known in the art and along withintegrated robotic systems and collections of chemical compounds/naturalproducts are extensively incorporated in high throughput screening sothat large numbers of test compounds can be tested for antagonist oragonist activity within a short amount of time. These methods includehomogeneous assay formats such as fluorescence resonance energytransfer, fluorescence polarization, time-resolved fluorescenceresonance energy transfer, scintillation proximity assays, reporter geneassays, fluorescence quenched enzyme substrate, chromogenic enzymesubstrate and electrochemiluminescence, as well as more traditionalheterogeneous assay formats such as enzyme-linked immunosorbant assays(ELISA) or radioimmunoassays.

[0149] Homogeneous assays are “mix and read” assays that are veryamenable to robotic application, whereas heterogeneous assays requireseparation of bound analyte from free by more complex unit operationssuch as filtration, centrifugation or washing. These assays are utilizedto detect a wide variety of specific biomolecular interactions and theinhibition thereof by small organic molecules, includingprotein-protein, receptor-ligand, enzyme-substrate, etc. These assaymethods and techniques are well known in the art and are described morefully in the following: High Throughput Screening: The Discovery ofBioactive Substances, John P. Devlin (ed.), Marcel Dekker, New York,1997, ISBN: 0-8247-0067-8; and the internet sites of lab-robotics.organd sbsonline.org. The screening assays of the present invention areamenable to high throughput screening of chemical libraries and aresuitable for the identification of small molecule drug candidates,antibodies, peptides and other antagonists and/or agonists.

[0150] One embodiment of a method for identifying molecules whichinhibit or antagonize the polypeptides involves adding a candidatemolecule to a medium which contains cells that express the polypeptidesof the instant invention; changing the conditions of said medium sothat, but for the presence of the candidate molecule, the polypeptideswould be bound to their natural ligands, substrates or effectormolecules, and observing the binding and stimulation or inhibition of afunctional response. The activity of the cells which were contacted withthe candidate molecule may then be compared with the identical cellswhich were not contacted and antagonists and agonists of thepolypeptides of the instant invention may be identified. The measurementof biological activity may be performed by a number of well-knownmethods such as measuring the amount of protein present (e.g. an ELISA)or of the proteins activity. A decrease in biological stimulation oractivation would indicate an antagonist. An increase would indicate anagonist.

[0151] Screening assays can further be designed to find molecules thatmimic the biological activity of the polypeptides of the instantinvention. Molecules which mimic the biological activity of apolypeptide may be useful for enhancing the biological activity of thepeptide. To identify compounds for therapeutically active agents thatmimic the biological activity of a polypeptide, it must first bedetermined whether a candidate molecule binds to the polypeptide. Abinding candidate molecule is then added to a biological assay todetermine its biological effects. The biological effects of thecandidate molecule are then compared to those of the polypeptide(s).

[0152] Another approach to development of therapeutic agents is peptidelibrary screening. The interaction of a protein ligand with its receptoroften takes place at a relatively large interface. However, asdemonstrated for human growth hormone and its receptor, only a few keyresidues at the interface contribute to most of the binding energy(Clackson et al., 1995, Science 267: 383-386). The bulk of the proteinligand merely displays the binding epitopes in the right topology orserves functions unrelated to binding. Thus, molecules of only “peptide”length (2 to 90 amino acids) can bind to the receptor protein or bindingpartner of even a large protein ligand such as a polypeptide of theinvention. Such peptides may mimic the bioactivity of the large proteinligand (“peptide agonists”) or, through competitive binding, inhibit thebioactivity of the large protein ligand (“peptide antagonists”).Exemplary peptide agonists and antagonists of polypeptides of theinvention may comprise a domain of a naturally occurring molecule or maycomprise randomized sequences. The term “randomized” as used to refer topeptide sequences refers to fully random sequences (e.g., selected byphage display methods or RNA-peptide screening) and sequences in whichone or more residues of a naturally occurring molecule is replaced by anamino acid residue not appearing in that position in the naturallyoccurring molecule. Phage display peptide libraries have emerged as apowerful method in identifying such peptide agonists and antagonists.See, for example, Scott et al., 1990, Science 249: 386; Devlin et al.,1990, Science 249: 404; U.S. Pat. Nos. 5,223,409; 5,733,731; 5,498,530;5,432,018; 5,338,665; 5,922,545; WO 96/40987; and WO 98/15833 (each ofwhich is incorporated by reference in its entirety). In such libraries,random peptide sequences are displayed by fusion with coat proteins offilamentous phage. Typically, the displayed peptides are affinity-elutedagainst an antibody-immobilized extracellular domain of a receptor. Theretained phages may be enriched by successive rounds of affinitypurification and repropagation. The best binding peptides may besequenced to identify key residues within one or more structurallyrelated families of peptides. The peptide sequences may also suggestwhich residues may be safely replaced by alanine scanning or bymutagenesis at the DNA level. Mutagenesis libraries may be created andscreened to further optimize the sequence of the best binders (Lowman,1997, Ann. Rev. Biophys. Biomol. Struct. 26: 401-424). Anotherbiological approach to screening soluble peptide mixtures uses yeast forexpression and secretion (Smith et al., 1993, Mol. Phannacol. 43:741-748) to search for peptides with favorable therapeutic properties.Hereinafter, this and related methods are referred to as “yeast-basedscreening.” A peptide library can also be fused to the carboxyl terminusof the lac repressor and expressed in E. coli. Another E. coli-basedmethod allows display on the cell's outer membrane by fusion with apeptidoglycan-associated lipoprotein (PAL). Hereinafter, these andrelated methods are collectively referred to as “E. coli display.” Inanother method, translation of random RNA is halted prior to ribosomerelease, resulting in a library of polypeptides with their associatedRNA still attached. Hereinafter, this and related methods arecollectively referred to as “ribosome display.” Other methods employpeptides linked to RNA; for example, PROfusion technology, Phylos, Inc.(see, for example, Roberts and Szostak, 1997, Proc. Natl. Acad. Sci. USA94: 12297-12303). Hereinafter, this and related methods are collectivelyreferred to as “RNA-peptide screening.” Chemically derived peptidelibraries have been developed in which peptides are immobilized onstable, non-biological materials, such as polyethylene rods orsolvent-permeable resins. Another chemically derived peptide libraryuses photolithography to scan peptides immobilized on glass slides.Hereinafter, these and related methods are collectively referred to as“chemical-peptide screening.” Chemical-peptide screening may beadvantageous in that it allows use of D-amino acids and other unnaturalanalogues, as well as non-peptide elements. Both biological and chemicalmethods are reviewed in Wells and Lowman, 1992, Curr. Opin. Biotechnol.3: 355-362.

[0153] In the case of known bioactive peptides, rational design ofpeptide ligands with favorable therapeutic properties can be completed.In such an approach, one makes stepwise changes to a peptide sequenceand determines the effect of the substitution upon bioactivity or apredictive biophysical property of the peptide (e.g., solutionstructure). Hereinafter, these techniques are collectively referred toas “rational design.” In one such technique, one makes a series ofpeptides in which one replaces a single residue at a time with alanine.This technique is commonly referred to as an “alanine walk” or an“alanine scan.” When two residues (contiguous or spaced apart) arereplaced, it is referred to as a “double alanine walk.” The resultantamino acid substitutions can be used alone or in combination to resultin a new peptide entity with favorable therapeutic properties.Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed (see, e.g.,Takasaki et al., 1997, Nature Biotech. 15: 1266-1270). Hereinafter,these and related methods are referred to as “protein structuralanalysis.” These analytical methods may also be used to investigate theinteraction between a receptor protein and peptides selected by phagedisplay, which may suggest further modification of the peptides toincrease binding affinity.

[0154] Peptide agonists and antagonists of polypeptides of the inventionmay be covalently linked to a vehicle molecule. The term “vehicle”refers to a molecule that prevents degradation and/or increaseshalf-life, reduces toxicity, reduces immunogenicity, or increasesbiological activity of a therapeutic protein. Exemplary vehicles includean Fc domain or a linear polymer (e.g., polyethylene glycol (PEG),polylysine, dextran, etc.); a branched-chain polymer (see, for example,U.S. Pat. Nos. 4,289,872; 5,229,490; WO 93/21259); a lipid; acholesterol group (such as a steroid); a carbohydrate or oligosaccharide(e.g., dextran); or any natural or synthetic protein, polypeptide orpeptide that binds to a salvage receptor.

[0155] Another embodiment of the invention relates to uses of Pellinopolypeptides to study cell signal transduction. Cellular signaling ofteninvolves a molecular activation cascade, during which a receptorpropagates a ligand-receptor mediated signal by specifically activatingintracellular kinases which phosphorylate target substrates. Thesesubstrates can themselves be kinases which become activated followingphosphorylation. Alternatively, they can be adaptor molecules thatfacilitate down stream signaling through protein-protein interactionfollowing phosphorylation. Accordingly, these novel Pellino polypeptidescan be used as reagents to identify novel molecules involved in signaltransduction pathways.

[0156] The inventive polypeptides are involved in IL-1 signaling, and assuch can be used as inhibitors of the IL-1 signaling pathway.Accordingly, they find utility in in vitro screening assays and in vivotherapeutics. As therapeutics that are cell membrane permeable, thePellino polypeptides and fragments thereof can be administered toagonize or antagonize IL-1R mediated signaling pathways, thus providinguseful immunoregulators. Various liposome-based compositions of theinventive polypeptides are envisioned herein.

[0157] Compositions of the present invention may contain a polypeptidein any form described herein, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble polypeptide or anoligomer comprising soluble Pellino polypeptides.

[0158] Compositions comprising an effective amount of a polypeptide ofthe present invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. The polypeptides can be formulated according to known methodsused to prepare pharmaceutically useful compositions. They can becombined in admixture, either as the sole active material or with otherknown active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in Remington's Pharmaceutical Sciences, 16th ed. 1980,Mack Publishing Company, Easton, Pa.

[0159] In addition, such compositions can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0160] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, or by inhalation. Theterm “parenteral” includes injection, e.g., by subcutaneous,intravenous, or intramuscular routes, also including localizedadministration, e.g., at a site of disease or injury (for example,intracoronary or intra tumor administration or injection into a jointundergoing an inflammatory reaction). Sustained release from implants isalso contemplated. One skilled in the pertinent art will recognize thatsuitable dosages will vary, depending upon such factors as the nature ofthe disorder to be treated, the patient's body weight, age, and generalcondition, and the route of administration. Preliminary doses can bedetermined according to animal tests, and the scaling of dosages forhuman administration is performed according to art-accepted practices.

[0161] The polypeptide of the instant invention may also be administeredby the method of protein transduction. In this method, the Pellinopolypeptide is covalently linked to a protein-transduction domain (PTD)such as, but not limited to, TAT, Antp, or VP22 (Schwarze et al., 2000,Cell Biology 10: 290-295). The PTD-linked Pellino polypeptides can thenbe transduced into cells by adding them to tissue-culture mediacontaining the cells (Schwarze et al., 1999, Science 285: 1569; Lindgrenet al., 2000, TiPS 21: 99; Derossi et al., 1998, Cell Biology 8: 84; WO00/34308; WO 99/29721; and WO 99/10376). Moreover, it has been foundthat DNA encoding a polypeptide can be administered to a mammal in sucha way that it is taken up by cells, and expressed. The resultant proteinwill then be available to exert a therapeutic effect. Accordingly,compositions comprising nucleic acids in physiologically acceptableformulations are also contemplated. DNA may be formulated for injection,for example.

[0162] Antibodies

[0163] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0164] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hindrances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

[0165] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0166] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

[0167] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide or a DNA encoding a polypeptide; harvestingspleen cells from the immunized animal; fusing said spleen cells to amyeloma cell line, thereby generating hybridoma cells; and identifying ahybridoma cell line that produces a monoclonal antibody that binds thepolypeptide. The monoclonal antibodies may be recovered by conventionaltechniques.

[0168] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (Proc. Natl. Acad. Sci. USA84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winterand Harris (TIPS 14:139, May, 1993). Procedures to generate antibodiestransgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825and 5,545,806 and related patents claiming priority therefrom, all ofwhich are incorporated by reference herein.

[0169] Antigen-binding fragments of the antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fv, Fab, and F(ab′)2 fragments, and single-chain antibodies such asscFv. Antibody fragments and derivatives produced by genetic engineeringtechniques are also provided.

[0170] In one embodiment, the antibodies are specific for thepolypeptides of the present invention and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0171] The antibodies of the invention can be used in assays to detectthe presence of the polypeptides or fragments of the invention, eitherin vitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

[0172] The following examples are provided to further illustrateparticular embodiments of the invention, and are not to be construed aslimiting the scope of the present invention.

Example 1

[0173] Identification of Pellino Nucleic Acid and Polypeptide Sequences

[0174] The polynucleotide sequences of ESTs isolated from murinedendritic cells identified two clones containing open reading frameswith a high degree of similarity to the Drosophila protein Pellino(Grosshans et al., supra). Appropriate flanking PCR primers weredesigned, and a novel nucleic acid was amplified from a murine cDNAlibrary and cloned; the nucleotide sequence and encoded amino acidsequence of this clone, which is called Pellino-1 (previously referredto as Conserved Inflammatory Signal Target-1), are shown in SEQ ID NO:1and SEQ ID NO:2, respectively. Human sequences from the high-throughputgenomic (HTG) and EST divisions of the public GenBank database werecompared with murine Pellino-1, and an open reading frame for the humanPellino-1 homolog was assembled. PCR primers were designed based uponthis human sequence, and a cDNA clone was isolated by PCR amplificationfrom a human dermal fibroblast cDNA library. The nucleotide and aminoacid sequence of this protein are shown in SEQ ID NO:3 and SEQ ID NO:4,respectively. Subsequently, Rich et al. published the coding and aminoacid sequences of “human Pellino” (GenBank Accession Numbers AJ278859and CAC04320, Aug. 23, 2000); the amino acid sequence of the “humanPellino” polypeptide is identical to that of human Pellino-1 (SEQ IDNO:4) except for a Ser to Phe substitution at position 11. Thedifference between the human Pellino and SEQ ID NO:4 amino acidsequences may represent a naturally occurring allelic variation betweennucleic acids encoding these amino acid sequences within the humanpopulation. Partial Pellino-1 amino acid sequences have also beenpublished in WO 2000/58350; EP 1 074 617; and WO 2001/09318.

[0175] By querying public EST databases, a portion of a second, relatedgene, referred to as Pellino-2, was identified in the mouse and human.Pellino-2 amino acid sequences are 80% identical to their respectivePellino-1 counterparts. Appropriate primers were designed, and murinePellino-2 DNA was cloned substantially as described for Pellino-1; thenucleotide and amino acid sequence of murine Pellino-2 is shown in SEQID NO:5 and SEQ ID NO:6, respectively. The predicted nucleotide andamino acid sequence of human Pellino-2 is shown in SEQ ID NOs:7 and 8.

[0176] A data set was received from Celera Genomics (Rockville, Md.)containing a listing of amino acid sequences predicted to be encoded bythe human genome. This data set was searched with a BLAST algorithm toidentify Pellino polypeptide sequences and several partial amino acidsequences were found that appeared to be related to a new human Pellinopolypeptide, Pellino-3. Comparison of these partial Pellino-3 amino acidsequences to genomic and cDNA sequences allowed the predicted humanPellino-3 nucleotide and amino acid sequences, SEQ ID NO:11 and SEQ IDNO:12, respectively, to be assembled. Two possible allelic variationshave been detected within the human Pellino-3 amino acid sequence (SEQID NO:12): a deletion of the Leu residue at position 96, and an Arg toAla substitution at residue 353.

[0177] The amino acid sequences of murine (“M{circumflex over (m)}”) andhuman (“Hs”) Pellino-1 and Pellino-2 polypeptides, and human Pellino-3polypeptide (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQID NO:12) were compared with the amino acid sequences of Pellino-relatedPellino polypeptides from other species (Drosophila melanogaster, “Dm”,SEQ ID NO: 13; Ciona intestinalis, “Ci”, SEQ ID NO:14; andCaenorhabditis elegans, “Ce”, SEQ ID NO:15) using the GCG “pretty”multiple sequence alignment program, with amino acid similarity scoringmatrix=blosum62, gap creation penalty=8, and gap extension penalty=2.The alignment of these sequences is shown in Table 1, and indicatesconsensus amino acid residues which are identical among at least threeof the amino acid sequences in the alignment. The capitalized residuesin the alignment are those which match the consensus residues. Thenumbering of residues in the alignment corresponds to the position ofamino acids within the murine and human Pellino-1 polypeptides (SEQ IDNOs 2 and 4, respectively). Pellino-1 and -2 share 82% identity at theamino acid level, and the degree of conservation between human and mouseis extremely high; only one amino acid is different between human andmouse Pellino-1, and Pellino-2 is 95% conserved between these species.The predicted Pellino-3 amino acid sequence is 70% and 71% identical tohuman Pellino-1 and -2, respectively. There is a surprising degree ofsimilarity between human Pellino-1, for example, and the homologousprotein from C. elegans (SEQ ID NO:15), which share 44% amino acididentity and 53% amino acid similarity. It is evident from a cursoryinspection of the alignment that sequence conservation is notconcentrated in any particular part of the Pellino protein, but extendsthroughout. This, and the fact that all the Pellino polypeptides (exceptfor Drosophila Pellino and human Pellino-3, which contain smallN-terminal extensions) are of a very similar size, suggest that (1) allparts of the protein are involved in its wild-type function and (2) fewor no amino acids extraneous to that wild-type function exist in thepolypeptides. There is a particularly well-conserved central domain,extending from amino acid 132 through amino acid 193 in Pellino-1 (SEQID NOs 2 and 4; which corresponds to amino acids 134 through 195 in SEQID NO:8 and amino acids 158 through 219 in SEQ ID NO:12), and anabsolutely conserved motif from residue 245 through residue 254 of SEQID NOs 2 and 4 (which corresponds to amino acids 247 through 256 in SEQID NO:8 and amino acids 271 through 280 in SEQ ID NO:12). The C-terminalportions of Pellino polypeptides are interspersed by a series of short,invariant motifs, in which cysteine, proline, histidine and largehydrophobic residues are prevalent. The arrangement of some of theconserved sequences, including a Cys-Gly-His triplet and twoCys-Pro-X-Cys motifs, is reminiscent of the structure of the C3HC4RING-finger subfamily of Zinc-finger domains, which mediateprotein-protein and protein-DNA interactions in a diverse group ofproteins, including tumor suppressors, proto-oncogenes, and signalingmolecules including the TRAFs, with specific examples of polypeptidescontaining similar RING-finger domains including human ring fingerprotein-1 (hRING1, GenBank NP_(—)002922); chicken ring finger protein(C-RZF, GenBank 1589724); human proto-oncogene CBL (hC-CBL, GenBankP22681); murine TNFR2-TRAF signaling complex protein (mc-IAP1, GenBankAAC42078); human TRAF-interacting protein (hTRIP, GenBank NP_(—)005870);human TNF receptor-associated factor 3 (hTRAF3, GenBank NP_(—)003291);human TNF receptor-associated factor 2 (hTRAF2, GenBank NP—066961); andDrosophila neuralized protein (neu, GenBank S35371). The PellinoRING-finger-like domains comprise the following amino acid sequences:amino acid 333 through amino acid 398 of SEQ ID NOs 2 and 4; amino acids335 through 400 in SEQ ID NO:8 and the corresponding region of SEQ IDNO:6; and amino acids 360 through 425 in SEQ ID NO:12. There areconserved cysteine residues within the Pellino polypeptideRING-finger-like domains, located at positions 333, 336, 367, 371, 395,and 398 of SEQ ID NOs 2 and 4 (and at positions 335, 338, 369, 373, 397,and 400 of SEQ ID NO:8 and the corresponding positions in SEQ ID NO:6,and at positions 360, 363, 394, 398, 422, and 425 of SEQ ID NO:12). InPellino polypeptides the conserved cysteine and histidine residues aremore widely separated than would be typical for a classical RING-fingerdomain, in which the intervening sequences form the finger-like loops.The first cysteine following the conserved histidine of the canonicalRING-finger domain is missing in Pellino, but we note that there is analmost invariant histidine at position 362 of SEQ ID NOs 2 and 4 (and atposition 364 of SEQ ID NO:8 and the corresponding position in SEQ IDNO:6, and at positions 389 of SEQ ID NO:12) which might be available forco-ordination to a metal ion. A second, invariant Cys-Gly-His triplet atresidues 311-313 of SEQ ID NOs 2 and 4 (and at amino acids 313 through315 of SEQ ID NO:8 and the corresponding residues in SEQ ID NO:6, and atamino acids 338 through 340 of SEQ ID NO:12) extends the zinc-fingerresemblance further toward the N-terminus. There is also a conservedCys-Pro-Val motif at amino acids 282-284 of SEQ ID NOs 2 and 4 (and atthe corresponding positions of the other Pellino sequences in Table 1).Therefore, the C-terminal region of Pellino polypeptides appears tocontain a novel type of Zinc finger-like domain.

[0178] Regions of amino acid similarity have also been identifiedbetween Pellino polypeptides and an insect pox virus gene, Melanoplussanguinipes Entomopoxvirus (MsEPV) ORF244, which is believed to play arole in circumventing host immune defenses by blocking host defensiveprotein interactions (Rich et al., 2000, Immunogenetics 52: 145-149).

[0179] Amino acid substitutions and other alterations (deletions,insertions, etc.) to the Pellino amino acid sequences (SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:12) are predicted to bemore likely to alter or disrupt Pellino polypeptide activities if theyresult in changes to the capitalized residues of the amino acidsequences as shown in Table 1, and particularly if those changes do notsubstitute a residue present in other Pellino polypeptides at thatposition in the alignment shown in Table 1. Conversely, if a change ismade to the Pellino amino acid sequence resulting in substitution of oneor more Table 1 consensus sequence residue(s) for the Pellino residue(s)at those positions, it is less likely that such an alteration willaffect Pellino polypeptide function. Embodiments of the inventioninclude Pellino polypeptides and fragments of Pellino polypeptidescomprising altered amino acid sequences. Altered Pellino polypeptidesequences share at least 30%, or more preferably at least 40%, or morepreferably at least 50%, or more preferably at least 55%, or morepreferably at least 60%, or more preferably at least 65%, or morepreferably at least 70%, or more preferably at least 75%, or morepreferably at least 80%, or more preferably at least 85%, or morepreferably at least 90%, or more preferably at least 95%, or morepreferably at least 97.5%, or more preferably at least 99%, or mostpreferably at least 99.5% amino acid identity with the Pellino aminoacid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,and SEQ ID NO:12. TABLE 1 Amino Acid Sequence Comparison between PellinoPolypeptides from Different Species SEQ ID NO: 1 17 Hs Pellino-1 4˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜MFSPdQEnH . . PSKaPVKY Mm Pellino-1 2˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜MFSPdQEnH . . PsKaPVKY Hs Pellino-2 8˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜MFSPGQEeH cAPnKEPVKY Mm Pellino-2 6˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜MFSPGQEep sAPnKEPVKY Hs Pellino-3 12˜˜˜˜mvlegn pevgsprtsd lqhrgnkgsc vlsSPGed . . aqPgeEPiKY Dm Pellino 13˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜m vkrtdgtesp ilaedGgdgH dkPr . . . lrY Ci Pellino 14mkqegmdvsa spalavaggm pmdiqfeaga syhnfsQEda pkedegdiiY Ce Pellino 15˜˜˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜˜˜˜ ˜mvdeselen gtpSPpaysn eAildddi.Y consensus---------- ---------- ---------- -MFSPGQE-H -AP-KEPVKY 18 65 HsPellino-1 4 GELIVLGYNG sLPNGDRGRR .KSRFALfKR PKANGVKPST VHIacTPQA. MmPellino-1 2 GELIVLGYNG sLPNGDRGRR .KSRFALfKR PKANGVKPST VHIacTPQA. HsPellino-2 8 GELvVLGYNG aLPNGDRGRR .KSRFALyKR PKANGVKPST VHviSTPQA. MmPellino-2 6 GELvVLGYNG aLPNGDRGRR .KSRFALyKR tyAsGVKPST iHmVSTPQA. HsPellino-3 12 GELIVLGYNG cLasGDkGRR .rSRlALSrR shANGVKPdv mHhiSTPlv. DmPellino 13 GELviLGYNG yLPqGDRGRR .rSkFvLhKR teAsGVKrSk hyIVqsPQt. CiPellino 14 GqLIVLGtNG qLPtGDkGRR .rScFtLrrk rKAtGVKPSd qHqVyqkash CePellino 15 GELIlLGfNG qaeNratskR yltekvLrrR dsANGiKkcT VHnVST . . sdconsensus GELIVLGYNG -LPNGDRGRR -KSRFAL-KR PKANGVKPST VHIVSTPQA- 66 115Hs Pellino-1 4 aKAISNKdQH SISYTLSRaQ TVVVEYTHDS nTDMFQIGRS TESPIDFVVT MmPellino-1 2 aKAISNKdQH SISYTLSRaQ TVVVEYTHDS nTDMFQIGRS TESPIDFVVT HsPellino-2 8 SKAIScKgQH SISYTLSRnQ TVVVEYTHDk dTDMFQvGRS TESPIDFVVT MmPellino-2 6 SKAISsrghH SISYTLSRsQ TVVVEYTHDk dTDMFQvGRS TESPIDFVVT HsPellino-3 12 SKAlSNrgQH SISYTLSRsh sViVEYTHDS dTDMFQIGRS TEnmIDFVVT DmPellino 13 SKAIldanQH SISYTLSRnQ aViVEYkeDt eTDMFQvGRS sESPIDFVVm CiPellino 14 SetflsKdhH SvSYTLpRs. vVVVpYvHDd nsDMFQIGRS TEePIDFVlm CePellino 15 tKltkdKarH tvSfhsdsnk sVViEYaaDp skDMFQIGRa sddqIDFtViconsensus SKAISNK-QH SISYTLSR-Q TVVVEYTHDS -TDMFQIGRS TESPIDFVVT 116 152Hs Pellino-1 4 DT . . . . VPGS . . . . QSnsDt QSvQ . . . . . STISRFACRIi CeRNpPfTAR Mm Pellino-1 2 DT . . . . VPGS . . . . QsnsDt QSvQ. . . . . S TISRFACRIi CeRspPfTAR Hs Pellino-2 8 DT . . . . isGS . . . .Qntdea QitQ . . . . . S TISRFACRIv CDRNePYTAR Mm Pellino-2 6 DT . . . .VsGg . . . . Qned.a QitQ . . . . . S TISRFACRIv CDRNePYTAR Hs Pellino-312 DT . . . . SPGg . . . . . gaaeg pSaQ . . . . . S TISRyACRIlCDRrpPYTAR Dm Pellino 13 DT . . . . lPGd . . . . kk . . Da kvmQ . . . .. S TISRFACRIl vnRcePakAR Ci Pellino 14 Di . . . . eaGS siptnhkpqt QpkQ. . . . . S TISRFACRIv CDRehPYTsR Ce Pellino 15 DTwmflpehS daavparpqidvlekgdrtS TISRFACRIl iDRensnkAy consensus DT----VPGS ----Q----D-QS-Q-----S TISRFACRI- CDRN-PYTAR 153 198 Hs Pellino-1 4 IYAAGFDSSKNIFLGEKAAK WKT.sDGq . . MDGLTTNGVL VMHPRnGFT. Mm Pellino-1 2 IYAAGFDSSKNIFLGEKAAK WKT.sDGq . . MDGLTTNGVL VMHPRnGFT. Hs Pellino-2 8 IfAAGFDSSKNIFLGvKAAK WKn.pDGh . . MDGLTTNGVL VMHPRGGFT. Mm Pellino-2 6 IfAAGFDSSKNIFLGEKAAK WKn.pDGh . . MDGLTTNGVL VMHPqGGFT. Hs Pellino-3 12 IYAAGFDaSsNIFLGErAAK WrT.pDGl . . MDGLTTNGVL VMHPaGGFs. Dm Pellino 13 IfAAGFDSSrNIFLGEKAtK Wqd . . nve . . iDGLTTNGVL iMHPkGsFcg Ci Pellino 14IYAAGFDtSm NIiLGEKApK WtTeqnGkki iDGLTTNGVL iMqPkncFs. Ce Pellino 15IYAAGFDahq NIsinkKslK W.TksnGe . . vDGLTTNGVL llHPnkddll consensusIYAAGFDSSK NIFLGEKAAK WKT--DG--- MDGLTTNGVL VMHPRGGFT-

[0180] The human Pellino-1, -2, and -3 coding sequences were comparedwith publicly available preliminary human genomic DNA sequences, and thefollowing chromosome 2, 14, and 11 contigs were identified as containinghuman Pellino-1, -2, and -3 coding sequences, respectively: AC013466.3(Pellino-1), AL138995.4 and AL355073.4 (Pellino-2), and AC027270.3(Pellino-3). The approximate positions of the exons containingPellino-1, -2, and -3 coding sequences in the above contigs are shown inthe table below, along with their locations relative to SEQ ID NOs 3, 7,and 11; note that the 5′ and 3′ untranslated regions may extend furtheralong the contig sequence beyond those portions that correspond to SEQID NOs 3, 7, and 11, as indicated by the parentheses around the contigendpoints in the table. Note also that the positions of exon boundariesare very highly conserved within the human Pellino-1, -2, and -3 codingsequences, with the differences in exon size primarily occurring in Exon1, with Pellino-2 having two additional codons in Exon 1 relative toPellino-1, and Pellino-3 having 27 additional codons in Exon 1 relativeto Pellino-1. Due to the preliminary sequence and assembly of the contigsequence, the exons within the contig are not always in the right orderor orientation with respect to each other, and may contain sequencevariations due to inaccurate sequence data or allelic polymorphism. Forexample, the genomic contig AC013466.3 has two copies of the Pellino-1Exon 2 sequence present in opposite orientations with respect to eachother, as indicated in the table below. Position of Pellino-1 Positionin exons in AC013466.3 SEQ ID NO:3 Exon 1 (32275)-32205    1-71 Exon 228794-28666;  72-201 74461-74589 Exon 3 78789-78890 202-303 Exon 482682-82879 304-501 Exon 5 82979-83167 502-690 Exon 6   84024-(84590) 691-1257 Position of Pellino-2 Position in exons in AL138995.4 SEQ IDNO:7 Exon 1 (81889)-81965    1-77 Exon 2 141562-141691  78-207 Positionof Pellino-2 Position in exons in AL355073.4 SEQ ID NO:7 Exon 381379-81480 208-309 Exon 4 90140-90337 310-507 Exon 5 91971-92159508-696 Exon 6   98303-(98869)  697-1263 Position of Pellino-3 Positionin exons in AC027270.3 SEQ ID NO:11 Exon 1 (16496)-16646    1-152 Exon 219608-19737 153-282 Exon 3 75962-76063 283-384 Exon 4 76834-77027385-579 Exon 5 77329-77521 580-772 Exon 6   79193-(79754)  773-1338

[0181] Corresponding positions of Pellino-1, -2, and -3 gene exons inhuman contigs and in cDNA sequences:

[0182] The genomic sequences comprising human Pellino-1 exons map to the2pl3.3 region of human chromosome 2. Human Pellino nucleic acids such asSEQ ID NO:3 and fragments thereof are useful for the cytologicalidentification of this chromosomal region, and for the genomic mappingof human genetic disorders such as the following disorders that havebeen mapped to this region: Preeclampsia/Eclampsia gene 1, AlstromSyndrome, Parkinson Disease gene 3, Orofacial Cleft gene 2, and WelanderDistal Myopathy. The genomic sequences comprising human Pellino-2 exonsmap to the 14q24.3 region of human chromosome 14. Human Pellino nucleicacids such as SEQ ID NO:7 and fragments thereof are useful for thecytological identification of this chromosomal region, and for thegenomic mapping of human genetic disorders such as the followingdisorders that have been mapped to this region: Achromatopsia gene 1,Hereditary Benign Chorea, Multinodular Goiter, Myopathy (Distal),Tyrosinemia Type 1B, and Alzheimer's Disease gene 3. The genomicsequences comprising human Pellino-3 exons map to a region of humanchromosome 11 between 11p11.1 and 11q13, and are believed to map mostclosely to the 11q12.1 region. Human Pellino nucleic acids such as SEQID NO:11 and fragments thereof are useful for the cytologicalidentification of this chromosomal region, and for the genomic mappingof human genetic disorders such as the following disorders that havebeen mapped to this region: Osteoporosis-Pseudoglioma Syndrome andSpinocerebellar Ataxia gene 5. The murine Pellino-1 gene is locatedwithin the same genetic region as the mouse Wobbler mutation (Fuchs etal., 2002, BMC Genetics 3: 14), which causes degeneration of spinalmotoneurons. It is intriguing that all three of the human Pellino genesmap near human genetic loci involving neuromuscular defects: WelanderDistal Myopathy; Myopathy (Distal); and Spinocerebellar Ataxia gene 5(which involves failure of muscular coordination and/or irregularity ofmuscular action), suggesting that these human genetic defects mayinvolve defects in human Pellino.polypeptide activity.

EXAMPLE 2

[0183] Reporter Gene Assays of Pellino Polypeptide Activity

[0184] The murine Pellino-1 coding sequence DNA was fused, in frame atthe 3′ end, to DNA encoding the FLAG epitope, followed by an in-framestop codon. This construct was cloned into the mammalian expressionvector pDC304 (identical to pDC302, described in U.S. Pat. No.5,599,905, issued Feb. 4, 1997, except that the early splice region,consisting of splice donor and acceptor sites of the SV40 viral element,has been removed); and transfected into an IL-1-responsive line of COS-7cells by the DEAE-dextran method.

[0185] In order to assay the effect of the Pellino-1-FLAG polypeptideand other forms of Pellino polypeptides on reporter gene activity, amethod essentially as described in Born et al.., 1998, J. Biol. Chem.273: 29445-29450 (which is incorporated by reference herein) can beused. As one example of this method, Cos7 cells were transientlytransfected by the DEAE-dextran method as described (Cosman et al.,1984, Nature 312: 768-771, which is incorporated by reference herein),using 150 ng of the Pellino polypeptide expression construct and 700 ngof the reporter plasmid per 45,000 cells. Two days post-transfection,cells were stimulated with 10 ng/ml IL-1 or 40 ng/ml IL-18 (PeproTech,Inc.) for 4 hours. Cells were lysed and luciferase activity assessedusing Reporter Lysis Buffer and Luciferase Assay Reagent (PromegaCorp.). IL-8 promoter-reporter and NF-kB-dependent reporter constructsmay be used as reporter plasmids. Alternatively, the effect of Pellinopolypeptide expression may be assayed in COS-1 cells. In an alternativepreferred method, COS7 cells were grown in 12-well dishes as describedabove. The cells were transiently transfected with Pellino test plasmid,50 ng of reporter plasmid DNA, and empty vector, as required, to a totalof 1 micrograms of total DNA per well. After 24 hours, stimulatingagents were added in a small amount (less than 0.5% final volume) ofmedium or dimethyl sulfoxide, and the cells were re-incubated for 5hours. Cells were lysed in luciferase Reporter Lysis Buffer (Promega,Madison Wis., 0.25 ml per well) and luciferase activity was measured ina EG&G/Berthold luminometer after addition of Luciferase Assay Reagent(Promega) according to the supplier's instructions. All results werenormalized to total protein content of the lysates as measured using themicro-BCA assay (Pierce, Rockford, Ill.).

[0186] In a preliminary experiment, expression of murine Pellino-1-FLAGin this manner was found to partially inhibit IL-1-inducedNF-kB-dependent reporter gene activity. This inhibitory effect ofPellino-1-FLAG may have been due to over-expression of the Pellinopolypeptide, as transfecting COS cells with high concentrations of aPellino-1- or Pellino-2-expressing construct has been demonstrated tohave an inhibitory effect on NF-kB-dependent reporter gene activity,possibly through the formation of homodimers or higher multimers ofPellino polypeptides that could have inhibitory effects in contrast tostimulatory effects of moderate concentrations of Pellino polypeptidemonomers. Other possibilities for the inhibitory effect of a preparationof Pellino-1-FLAG on NF-kB-dependent reporter gene activity are thepresence of mutated forms of the polypeptide as described below, or therelative presence or absence of a yet-to-be-characterized factor in aparticular cell line.

[0187] However, in later experiments with a murine Pellino-1-FLAGconstruct that was confirmed to comprise a wild-type Pellino-1 aminoacid sequence, the wild-type Pellino-1-FLAG polypeptide had astimulatory effect on IL-1-induced NF-kB-dependent reporter geneactivity (see Table 2, below); wild-type Pellino-1-FLAG also moderatelyaugmented Jun N-terminal kinase, p38 kinase, and ERK signaling mediatedby IL-1. When expressed in COS-1 cells, wild-type Pellino-1 polypeptidestimulates IL-8 promoter-reporter gene activity and NF-kB-dependentreporter activity in both the presence and absence of treatment withTNF-alpha, as compared to a vector-only control. In similar experiments,transfection of COS cells with moderate amounts of construct expressingwild-type Pellino-2 polypeptide also stimulates NF-kB-dependent reportergene activity.

[0188] To define regions of Pellino that determine its response topro-inflammatory mediators, a number of mutant Pellino expressionvectors were constructed. The apparent Mr of FLAG-Pellino-1 on SDS-PAGEgels is close to the value of 47,224 daltons calculated from the primarysequence, indicating a lack of extensive post-translationalmodification. It is therefore possible to predict the approximate pointin the primary sequence of Pellino-1 where cleavage should occur inorder to generate a 30-kDa N-terminal product. The closest residue tothis theoretical point is Phe-158; cleavage of the peptide bondpreceding this residue would result in a polypeptide with a mass of30,044 daltons. This region of Pellino-1 polypeptide was thereforechosen for mutation, since it might be expected that some of theresulting mutants would be resistant to cleavage, or otherwise alteredin their response to pro-inflammatory stimuli. Cleavage of Pellino issensitive to a chymotrypsin inhibitor, TPCK, and chymotrypsin has arequirement for a large, aromatic residue on one side of its cleavagesite. Reasoning that the Pellino-cleaving enzyme might share the samespecificity determinants, we chose to mutate four of the aromatic aminoacids that are invariably found in this region in the mammalian Pellinopolypeptides. The two internal deletion mutants were chosen to flank thepredicted cleavage site, and also to include highly conserved residues.In addition, a series of truncation mutants, deleting residues from bothamino- and carboxyl-termini, and mutants lacking conserved cysteineresidues in the RING-finger-like domain, were constructed as follows. Tomake a series of N-terminal deletions, short sense-strandoligonucleotide primers were synthesized in which a sequence containinga KpnI restriction site and a methionine codon was fused to murinePellino-1sequences beginning with the codons for Gly-51, Phe-100,Asp-181, and Val-231. These were used with an antisenseFLAG-BglII-adapted primer to amplify PCR fragments from murinePellino-1-FLAG template DNA. These fragments were re-cloned into pDC304vector to generate, respectively, the constructs encoding the dN50-FLAG,dN99-FLAG, dN180-FLAG, and dN230-FLAG mutants. A similar strategy wasused to construct a series of mutants progressively truncated at theC-terminus; antisense oligonucleotides terminating with the codons forThr-150, Thr-250, and Glu-350 were synthesized in tandem with a sequencecontaining a stop codon and a BglII restriction site. These primers wereused to generate PCR fragments which were subsequently cloned intopDC304 and referred to as encoding the 1-150, 1-250, and 1-350 mutants,respectively. The constructs encoding the single amino-acidsubstitutions F137L-FLAG, Y154A-FLAG, F158A-FLAG, and F165L-FLAG wereconstructed using the QuickChange site-directed mutagenesis kit(Stratagene, LaJolla, Calif.). To construct the internal deletionmutants, d133-156-FLAG and d155-158-FLAG, we made, in each case, a pairof PCR fragments containing a restriction site introduced to flank thesequence to be deleted. Following restriction digestion and purificationof the PCR fragment pairs, they were three-way ligated into pDC304vector. A mutant was also constructed in which four RING-finger-likedomain amino acids from Cys-333 through Cys-336 were replaced by thetwo-amino-acid sequence Gly-Ser; this mutant is referred to asC333-C336GS-FLAG.

[0189] In contrast to the effect of wild-type Pellino-1, mutant forms ofPellino-1 polypeptides with amino acids 133-156 or amino acids 155-158of SEQ ID NO:2 deleted, or with 50 or 99 amino acids of the N-terminalregion of the polypeptide deleted, or with substitutions in theRING-finger-like domain that remove cysteine residues, inhibited IL-8promoter-reporter gene activity in both the presence and absence oftreatment with TNF-alpha, and the mutant form of Pellino-1 with aminoacids 155-158 deleted inhibited NF-kB-dependent reporter activity inboth the presence and absence of treatment with PMA, as compared to avector-only control. Stimulation of the activity of these reporter genesis consistent with a stimulatory effect on a pro-inflammatory regulatorycascade, while inhibition of the activity of these reporter genes isconsistent with an inhibitory effect on a pro-inflammatory regulatorycascade. A summary of the effects of wild-type and mutant forms ofmurine Pellino-1 on NF-kB-dependent reporter gene activity is summarizedin Table 2; all amino acid positions are in reference to the amino acidsequence of SEQ ID NO:2. The “Soluble or Insoluble” results aredescribed in Examples 3 and 4 below.

[0190] It can be seen from the Table below that deleting some of theN-terminal region of the Pellino-1 polypeptide (i.e. deleting theN-terminal 50 or 99 amino acids) generates mutants having inhibitoryactivity on MAP kinase-activated signaling pathways (as demonstrated forexample by inhibition of NF-kB-dependent transcription), but deletingmore substantial portions (i.e. the N-terminal 180 or 230 amino acids)abolishes the ability of Pellino-1 to stimulate or to inhibit reportergene activity. Therefore, it should be possible to make additionalN-terminal deletion mutants of Pellino polypeptides having inhibitoryactivity on NF-kB-dependent transcription, for example, a mutant inwhich N-terminal amino acids corresponding to the 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 N-terminalamino acids of SEQ ID NO:2 are deleted, but which still retainsinhibitory activity. Alternatively, other residues within the N-terminalamino acids of Pellino polypeptides corresponding to the 180 N-terminalamino acids of SEQ ID NO:2 may be deleted in order to generate mutantforms of Pellino polypeptides having inhibitory activity onNF-kB-dependent transcription. Similarly, mutants in which deletions (ofone to 50 amino acids and more preferably one to 30 mino acids) are madewithin the central conserved domain and the RING-finger-like domain mayalso exhibit inhibitory activity on NF-kB-dependent transcription. Allsuch mutants can be readily tested for activity using the reporter geneassays described herein. TABLE 2 Effect on Soluble or Clea- ReporterForm of Pellino-1 Description Insoluble ved? Gene wild-type-FLAGunaltered Pellino-1 Soluble + Stimulatory d133-156-FLAG amino acids 133-Insoluble + Inhibitory 156 deleted d155-158-FLAG amino acids 155-Insoluble + Inhibitory 158 deleted dN50-FLAG N-terminal 50 Insoluble +Inhibitory amino acids deleted dN99-FLAG N-terminal 99 Insoluble +Inhibitory amino acids deleted dN180-FLAG N-terminal 180 Insoluble n/ainactive amino acids deleted dN230-FLAG N-terminal 230 Insoluble n/ainactive amino acids deleted 1-250 only amino acids Insoluble + inactive1-250 present 1-350 only amino acids Soluble + inactive 1-350 presentF137L-FLAG Phe to Leu substi- Soluble + not tested tution at residue 137Y154A-FLAG Tyr to Ala substi- Soluble +/− Stimulatory tution at residue154 F158A-FLAG Phe to Ala substi- Soluble + Slightly tution at residue158 Stimulatory F165L-FLAG Phe to Leu substi- Soluble − Stimulatorytution at residue 165 C333-C336GS- replacement of not not InhibitoryFLAG Cys-333 through tested tested Cys-336 with Gly-Ser

[0191] In similar experiments using COS cells transfected with areporter construct including CHOP, a p38-dependent promoter, thed155-158-FLAG mutant form of Pellino-1 inhibited TNF-alpha stimulationof the CHOP reporter gene activity. This result is significant becauseit demonstrates that mutant forms of Pellino polypeptides are able toinhibit multiple MAP kinase-activated pro-inflammatory signalingpathways, as indicated by their inhibition of both NF-kB-dependenttranscription and p38-dependent transcription. Because wild-type formsof Pellino polypeptides have stimulatory effects on key components ofthe four major MAP kinase-activated signaling pathways—stimulation ofJun N-terminal kinase, p38 kinase, and ERK signaling, and stimulation ofNF-kB-dependent transcription—the “dominant-negative” mutant forms ofPellino polypeptides are expected to inhibit the Jun kinase and ERK MAPkinase-activated signaling pathways in a similar fashion to theinhibition of the p38 and NF-kB MAP kinase-activated signaling pathways.

EXAMPLE 3

[0192] Intracellular Localization of Pellino Polypeptides

[0193] This example describes a method for monitoring the regulation byIL-1 (or other cytokine or molecule) of the intracellular localizationof Pellino-1 in cells. COS-7 cells (which express an endogenous IL-1receptor) are transfected with an expression vector comprisingFLAG-Pellino-1 as described in Example 2. Cells may also be transfectedat the same time with other cDNAs encoding proteins which mediateinflammatory signaling, such as IL-1 Receptor-associated kinase (IRAK;GenBank NP001560). Transfected cells are cultured for about 48 hours.IL-1 (20 ng/ml), or another cytokine or molecule at an appropriateconcentration, is added to the culture medium for the last 15 minutes(short-term stimulation) or 24 hours (prolonged stimulation) of thisculture period.

[0194] The cell cultures are washed with ice-cold phosphate-bufferedsaline (PBS), and cell lysates are prepared by scraping the cells intolysis buffer (a buffer containing 50 mM Tris-chloride pH 8.0supplemented with 1% nonidet (NP-40), 0.5% sodium deoxycholate, 0.1 mMsodium orthovanadate, 30 mM para-nitrophenol phosphate, 30 mMbeta-glycerophosphate, 140 mM NaCl, 5 M dithiothreitol, 2 mM EDTA, 10 mMleupeptin, 10 mM pepstatin A and 1 mM phenymethylsulfonyl fluoride;chemicals purchased from Sigma, St. Louis, Mo.). Solubilization of thecellular proteins can be facilitated by passage of the lysates severaltimes through 25-gauge hypodermic needles. The lysate is centrifuged at13,000×G at 4° C. The supernatant at this stage is referred to as the“soluble fraction.” The remaining pellet is solubilized in 1× SDS-PAGEsample buffer (Laemmli et al., Nature 227:680; 1970); this material isreferred to as the “insoluble fraction.”

[0195] In an alternate, preferred, procedure for obtaining soluble andinsoluble fractions comprising Pellino polypeptides, COS7 (monkeykidney) cells were maintained in Dulbecco's modified Eagles mediumcontaining 5% fetal bovine serum and supplemented with 100 units/mlpenicillin and 100 micrograms/ml streptomycin at 35° C. in 5% CO₂.Plasmids encoding Pellino-FLAG, or other expression plasmids, weretransiently transfected into confluent COS7 cells in 6-well tissueculture dishes (Costar) using DEAE-dextran. At various times aftertransfection, cells were scraped into 0.4 ml of an lysis/extractionbuffer consisting of 50 mM Tris-HCl pH 7.8, 1% NP-40, 0.15M NaCl, 2 mMEGTA, 5 microM NaF, 30 mM β-glycerophosphate, 1 mM sodium orthovanadate,1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10micrograms/ml leupeptin, and 10 micrograms/ml pepstatin A. The cellsuspensions were allowed to lyse on ice for 15 minutes and werecentrifuged (13000 rpm for 10 minutes at 4 degrees C.) in amicrocentrifuge. The supernatants were carefully removed to fresh tubesand diluted with one-third volume of 4×-concentrated SDS-PAGE samplebuffer containing 2-mercaptoethanol. The supernatant samples(‘detergent-soluble’ fraction) were boiled for 5 minutes. The pelletswere re-extracted by resuspending them in one half the original volumeof 1×-concentrated SDS-PAGE sample buffer, vortexing, and then boilingthem.

[0196] Matched aliquots of soluble fraction and insoluble fraction maythen be analyzed by gel electrophoresis on 4-20% gradientSDS-polyacrylamide gels (Novex, Invitrogen Corp., Carlsbad Calif.), andtransferred to nitrocellulose membranes, and assayed by westernimmunoblotting using anti-FLAG antibodies (i.e., FLAG M2) to bind to theprotein products of the transfected cDNAs. Proteins are visualized byincubation of western blots with horseradish-peroxidase-conjugatedanti-mouse IgG (BioRad, San Diego, Calif.) followed by detection usingthe ECL system (Amersham; Arlington Heights, Ill.).

EXAMPLE 4

[0197] Effect of Cell Stimulation on Pellino Polypeptide Localizationand Cleavage

[0198] This example describes the effect of stimulation of COS-7 cellstransfected with an expression vector encoding FLAG-Pellino-1 with IL-1or other molecules. Transfected cells are prepared substantially asdescribed above. In some instances, the cells are co-transfected with apDC304 vector containing a cDNA insert coding for human IRAK with atandem 3′ FLAG and poly-His ‘tail.’ In further instances, the cells areco-transfected with a catalytically inactive human FLAG-polyHis-IRAKexpression vector in which lysine residue 293 in the ATP-binding pocketof IRAK is replaced with an alanine. As Drosophila Pellino wasidentified by its ability to associate with Pelle, experiments in whichPellino is coexpressed with IRAK have been performed to determine ifmammalian Pellino interacted with IRAK, the presumptive mammaliancounterpart of Pelle.

[0199] Analysis indicated that in the absence of over-expressed, activeIRAK, FLAG-Pellino-1 is largely present in the soluble fraction as apolypeptide close to the predicted size of 46 kDa. When IRAK is overexpressed, FLAG-Pellino-1 is largely found in the insoluble fraction,and a significant portion of the Pellino in the insoluble fractionappeared on Western blots as a 30-kDa species. Since it was reactivewith anti-Flag antibody, the 30-kDa species presumably consists of anamino-terminal fragment. In some experiments, in which larger amounts ofexpression plasmid cDNA were used, an additional Pellino N-terminalcleavage product of 17 kDa was detected. The 30-kDa insoluble form ofPellino-1 present in cells transfected with wild-type IRAK migratedslightly slower than that from cells co-transfected with kinase-inactiveIRAK, which might indicate that the 30-kDa Pellino-1 fragment wasdifferently phosphorylated, or perhaps modified in some other way.Similarly, there was an overall shift in the mobility of wild-type IRAKevident in cells which were co-transfected with Pellino-1, consistentwith a model in which both kinase-active IRAK and Pellino are involvedin the regulation of the state of the other's post-translationalmodifications (modifications such as phosphorylation, ubiquitinylation,myristoylation, farnesylation, and geranylgeranylation), butkinase-inactive IRAK can neither affect modifications to Pellino-1 norbe affected in this way by Pellino-1. In contrast, the cleavage ofPellino-1 and relocation to the insoluble fraction do not require thekinase activity of IRAK.

[0200] To determine if the observed redistribution and proteolyticprocessing of Pellino-1 specifically required IRAK,Pellino-1-transfected cells were stimulated with agents that activateNF-kB by IRAK-independent pathways such as phorbol myristate acetate(PMA, 100 ng/ml), which is known to promote many of the sameintracellular signals as IL-1, and TNF-alpha (20 ng/ml). TNF-alphaactivates NF-kB through a mechanism involving the adaptor protein TRADD,the kinase RIP, and TRAF2, while PMA stimulates the activity of proteinkinase C which is known to cross-talk with the NF-kB pathway. Bothagents caused a time-dependent increase in the redistribution ofPellino-1 into the insoluble fraction, where it was mostly present asthe 30-kDa cleavage product. In both cases, 2 to 3 hours of exposure tothe stimulus was required before significant Pellino cleavage was seen.The total amount of detectable Pellino-1 polypeptide (the sum of solubleand insoluble fractions) was increased in the presence of PMA orTNF-alpha, which presumably reflects a change in the net rates ofPellino-1 synthesis and/or degradation. However, incubation with variousgrowth factors (epidermal growth factor (EGF), basic fibroblast growthfactor, transforming growth factor-beta, or platelet-derived growthfactor) had little or no effect on Pellino-1; only epidermal growthfactor was very weakly active. EGF has been reported in some cells toactivate NF-kB through the induction of IkB-alpha degradation. Theseresults are consistent with Pellino-1 cleavage and redistributionoccurring specifically in response to stimuli which activate NF-kB,including those with signaling mechanisms not involving IRAK..Time-course experiments demonstrated that the cleavage and relocation ofPellino-1 began at about two hours after induction with PMA, and atabout three hours after induction with TNF-alpha, whereasTNF-alpha-mediated NF-kB activation is maximal in COS7 cells after 15minutes, suggesting that cleavage and changes in the solubility ofPellino-1 are more likely to be part of the cellular effects of NF-kBactivation than to be causally involved in the activation process, andindicating that Pellino-1 cleavage and relocation might depend upon denovo protein synthesis. This observation was supported by experiments inwhich pretreatment of FLAG-Pellino-1-transfected cells with the proteinsynthesis inhibitor cycloheximide largely prevented the ability of PMAto subsequently induce Pellino-1 cleavage and relocation. The slowkinetics of Pellino-1 processing would be consistent with a requirementfor de novo protein synthesis, consistent with this, treatment of COS7cells with the protein synthesis inhibitor cycloheximide completelyprevented its PMA-induced cleavage. It is therefore possible that PMAinduces the synthesis of a proteinase which cleaves Pellino, or thesynthesis of some accessory factor for a constitutively-expressedproteinase. For a 30 kDa Pellino-1 fragment to be generated, proteolyticcleavage would be predicted to occur within the region of amino acids132 to 189 of SEQ ID NO:2. Examination of the amino acid sequence ofPellino-1 shows it has been extremely well conserved in those speciesfor which EST sequence data is available (murine and human Pellino-1 andPellino-2, disclosed herein; Drosophila pellino, GenBank accessionnumber AF091624; and the F25B4.2 gene product of Caenorhabditis elegans,GenBank accession number U64842). A number of conserved phenylalanineand tyrosine residues are present in this region, any of which mightserve as the recognition site for a chymotrypsin-like serine protease.

EXAMPLE 5

[0201] GST-Pellino Fusion Polypeptides

[0202] This example describes the construction and expression of arecombinant Glutathione S-transferase (GST)-Pellino-1 fusion protein.PCR primers were synthesized having the sequencesATATTCACTGAATTCTGATGTTTTCTCCTGATCAA (Primer 1; SEQ ID NO:9) andAGTGAATATGAATTCCTACTTATCATCGTCATCTTTG (Primer 2; SEQ ID NO:10), for thesense and antisense primers, respectively. These were designed for usewith a Pellino-1-FLAG template, and add a EcoR1 site to each end of theamplified product. This PCR product was ligated into the unique EcoR1cloning site of the vector pGEX2T (described in EP 0293249-A) such thatthe coding sequences of the glutathione S-transferase gene andFLAG-Pellino-1 were in the same frame. E coli strain DH10B weretransformed with the resultant vector and a one-liter culture was grown.Transcription of the GST-Pellino-1-FLAG gene was induced by addition ofIPTG (0.1 mM) to the bacterial culture for three hours. Bacterial cellswere harvested and lysed according to methods well known in the art(see, for example, Smith D. B., Johnson K. S.; Gene 67:31-40(1988)). Thelysate containing solubilized GST-Pellino-1-FLAG was purified on 1 ml ofGlutathione-Agarose beads (Pharmacia), according to the directionssupplied by the manufacturer.

EXAMPLE 6

[0203] Anti-Pellino Monoclonal Antibodies

[0204] This example illustrates the preparation of monoclonal antibodiesagainst Pellino polypeptides. Preparations of purified recombinantPellino polypeptides, for example, or transfected cells expressing highlevels of Pellino polypeptides, are employed to generate monoclonalantibodies against Pellino polypeptides using conventional techniques,such as those disclosed in U.S. Pat. No. 4,411,993. DNA encoding Pellinopolypeptides can also be used as an immunogen, for example, as reviewedby Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies arelikely to be useful as components of diagnostic or research assays forPellino or Pellino activity, or in affinity purification of Pellinopolypeptides.

[0205] To immunize rodents, Pellino immunogen (for example, a Pellino-1peptide comprising amino acids 2 through 20, amino acids 118 through131, or amino acids 318 through 340 of SEQ ID NOs 2 and 4 preferablycoupled to an immunogenic molecule such as keyhole limpet hemocyanin, isemulsified in an adjuvant (such as complete or incomplete Freund'sadjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi,Hamilton, Mont.), and injected in amounts ranging from 10-100 microgramssubcutaneously into a selected rodent, for example, BALB/c mice or Lewisrats. DNA may be given intradermally (Raz et al., Proc. Natl. Acad. Sci.USA 91:9519, 1994) or intramuscularly (Wang et al., Proc. Natl. Acad.Sci. USA 90:4156, 1993); saline has been found to be a suitable diluentfor DNA-based antigens. Ten days to three weeks days later, theimmunized animals are boosted with additional immunogen and periodicallyboosted thereafter on a weekly, biweekly or every third weekimmunization schedule.

[0206] Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

[0207] Hybridoma clones thus generated can be screened by ELISA forreactivity with Pellino polypeptides, for example, by adaptations of thetechniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and inU.S. Pat. No. 4,703,004. A preferred screening technique is the antibodycapture technique described by Beckman et al., J. Immunol. 144:4212(1990). Positive clones are then injected into the peritoneal cavitiesof syngeneic rodents to produce ascites containing high concentrations(>1 mg/ml) of anti-Pellino monoclonal antibody. The resulting monoclonalantibody can be purified by ammonium sulfate precipitation followed bygel exclusion chromatography. Alternatively, affinity chromatographybased upon binding of antibody to protein A or protein G can also beused, as can affinity chromatography based upon binding to Pellinopolypeptide.

EXAMPLE 7

[0208] Northern Blot Analysis of Pellino-1 Expression

[0209] A 234-bp PCR fragment corresponding to the predicted 3′ end ofthe human Pellino-1 mRNA was generated by standard amplificationmethods. The PCR fragment was purified (Qiagen PCR purification kit) andlabeled by the Gibco Random Prime Oligonucleotide DNA Labeling kit. ThecDNA riboprobe was denatured at 100 degrees C. for 5 minutes and placedon ice. The cDNA probe was denatured at 100 degrees C. for 5 minutesbefore being added to the hybridization solution. A multi-tissuenorthern blot containing RNA from human tissues—brain, heart, skeletalmuscle, colon (no mucosa), thymus, spleen, kidney, liver, smallintestine, placenta, lung, and peripheral blood leukocytes—was purchasedfrom Clonetech (Palo Alto, Calif.). The blot was blocked in 5 mLExpressHyb Solution for 30 minutes at 68 degrees C. Fresh ExpressHybsolution containing the denatured radiolabeled cDNA probe was added tothe membrane and incubated at 68 degrees C. for 1 hour with continuousshaking. The blot was rinsed in 2×SSC, 0.05% SDS with four changes atroom temperature for 40 minutes, followed by a wash in 0.1×SSC, 0.1% SDSwith two changes for 40 minutes at 50 degrees C. The riboprobe forPellino-1 hybridized to the Northern blot with a major band at 4.4kilobases in all tissues represented, with the highest level ofexpression evident in peripheral blood leukocytes; moderate levels ofexpression in lung, placenta, liver, kidney, skeletal muscle, spleen,thymus, and brain; and low levels of expression in small intestine,colon, and heart. The message appears to be more highly expressed inperipheral blood leukocytes, and in this tissue there seems to be twoadditional bands at 7.5 and 9.5 kb which do not appear in the lanes forthe other tissues.

EXAMPLE 8

[0210] Pellino-1 Interacts with IRAK, IRAK-4, and TRAF6 in an IL-1Signaling Pathway Complex

[0211] Biochemical and genetic studies have postulated a model for theIL-1-mediated signaling pathway (FIG. 1). Upon IL-1 stimulation, theadaptor molecules MyD88 and Tollip are recruited to the IL-1 receptorcomplex, which then recruits IRAK4 and IRAK. IRAK ishyperphosphorylated, mediating the recruitment of TRAF6 to the receptorcomplex (Complex I). IRAK-TRAF6 then leaves Complex I to interact withpre-associated TAK1, TAB1, and TAB2 on the membrane, resulting in theformation of IRAK-TRAF6-TAK1-TAB1-TAB2 (Complex II). The formation ofComplex II leads to the phosphorylation of TAK1 and TAB2, whichfacilitates the dissociation of TRAF6-TAK1-TAB1-TAB2 (Complex III) fromIRAK and consequent translocation of Complex III to the cytosol. Theformation of Complex III and its interaction with additional factors inthe cytosol lead to the activation of TAK1. Activation of TAK1 leads tothe activation of both IKK and MKK6, resulting in activation of NF-kBand JNK, respectively. Phosphorylated IRAK remains on the membrane andeventually is ubiquitinated and degraded.

[0212] We examined whether mammalian Pellino-1 interacts with IRAK andIRAK4, which are the mammalian counterparts of Pelle. Since IRAK andIRAK4 are the essential signaling components of the IL-1 pathway, theinteraction of Pellino-1 with IRAK and IRAK4 was studied upon IL-1stimulation. 293 cells were transfected with flag-tagged Pellino-1 asfollows: 6×10⁵ cells were seeded onto each 15-cm plate and transfectedthe following day by the calcium phosphate method with 15 micrograms ofvector encoding flag-tagged Pellino-1. After 48 hours, cells were eitherleft untreated or were treated with 100 units/ml of recombinant humanIL-1 beta (National Cancer Institute), then lysed in a Triton-containinglysis buffer (0.5% Triton X-100, 20 mM HEPES pH 7.4, 150 mM NaCl, 12.5mM beta-glycerophosphate, 1.5 mM MgCl₂, 10 mM NaF, 2 mM dithiothreitol,1 mM sodium orthovanadate, 2 mM EGTA, 20 microM aprotinin, 1 mMphenylmethylsulfonyl fluoride). The cell extracts were thenimmunoprecipitated by incubation with 1 microgram of either anti-flag M2antibody (Sigma-Aldrich Corp., St. Louis, Mo.) or preimmune serum(negative control) for two hours, followed by a two-hour incubation with20 microliters of protein A-Sepharose beads (pre-washed and resuspendedin phosphate-buffered saline at a 1:1 ratio). After incubation the beadswere washed four times with lysis buffer, separated by SDS-PAGE,transferred to Immobilon-P membranes (Millipore, Bedford, Mass.), andanalyzed by Western immunoblotting using antibodies against IRAK andIRAK4 (FIG. 2, Panel A). Anti-IRAK was obtained from Santa CruzBiotechnologies (Santa Cruz, Calif.); and anti-IRAK4 was a gift from Dr.Holger Wesche (Tularik, South San Francisco, Calif.). Interestingly,Pellino-1 indeed forms an IL-1-dependent signaling complex with bothIRAK and IRAK4, strongly suggesting that Pellino-1 plays a role in anIL-1-mediated signaling pathway (FIG. 2, Panel A). To address themolecular mechanism by which Pellino-1 functions in IL-1 signaling, weinvestigated whether Pellino-1 interacts with IRAK and IRAK4 in thereceptor complex (Complex I) or in the TAK1-containing Complex II. Cellextracts prepared from 293 cells transfected with flag-tagged Pellino-1,either untreated or stimulated with IL-1beta, were immunoprecipitatedwith anti-flag M2 and anti-TRAF6 antibodies, followed by immunoblottingwith antibodies against IRAK, TRAF6,IL-1 receptor, TAK1, and TAB2 (FIG.2, Panel B). Anti-TRAF6 antibodies were obtained from Santa CruzBiotechnologies (Santa Cruz, Calif.), and rabbit anti-TAK1, anti-TAB1,and anti-TAB2 polyclonal antibodies were kindly provided by Dr. KunihiroMatsumoto (Ninomiya-Tsuji, J. et al., 1999, Nature 398, 252-256;Takaesu, G. et al., 2000, Mol Cell 5, 649-658). TRAF6 is present both inthe receptor complex (interacting with IL-1and IRAK) and the TAK1complex (interacting with TAK1 and TAB2). However, while Pellino-1formed a complex with IRAK, IRAK4, and TRAF6 (FIG. 2), itco-immunoprecipitated with neither the IL-1 receptor nor the TAK1complex (TAK1 and TAB2) (FIG. 2, Panel B), suggesting that theIL-1-induced Pellino-1-IRAK4-IRAK-TRAF6 complex is likely to exist as anintermediate between the receptor complex (Complex I) and theTAK1-containing complex (Complex II) (FIG. 1).

[0213] Since most of the signaling components in the IL-1-mediatedpathway are able to constitutively activate NF-kB upon overexpression,Pellino-1 was examined for this activity using the followingluceriferase reporter assay. IL-1-unresponsive mutant cells I1A and 13A(Li et al., 2001, Proc Natl Acad Sci USA 98: 4461-4465) were maintainedin Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetalcalf serum, penicillin G (100 micrograms/ml), and streptomycin (100micrograms/ml). 2×10⁵ 293 cells (“wild-type”), or mutant I1A or I3Acells, were seeded onto a 10-cm plate and cotransfected the followingday by the calcium phosphate method with 1 microgram of theNF-kB-dependent E-selectin-luciferase reporter plasmid pE-selectin-luc(Schindler and Baichwal, 1994, Mol Cell Biol 14: 5820-5831), 1 microgramof pSV₂-beta-gal, and either a given amount of Pellino-1 in a mammalianexpression vector, or 100 ng of the Pellino-1 expression vector incombination with a given amount of a vector expressing adominant-negative kinase-inactive TAK1 mutant (TAK1 DN, a kind gift fromDr. Kunihiro Matsumoto of Nagoya University, Japan). After 48 hours, thecells were split onto two 35-mm plates and, the next day, werestimulated with IL-1 as described above for four hours before harvest.Luciferase and beta-galactosidase activities were determined by usingthe luciferase assay system and chemiluminescent reagents from Promega(Madison, Wis.). Pellino-1 can activate the E-selectin promoter activityin a dose-dependent manner as shown in Table 3 below. We have previouslytaken a genetic approach to study IL-1-dependent signaling pathways,through random mutagenesis generating IL-1-unresponsive cell lineslacking specific components of the pathways. While mutant cell line I1Alacks both IRAK protein and mRNA, mutant I3A is defective in a componentupstream of IRAK but downstream of the IL-1 receptor (Li et al., 1999,Mol Cell Biol 19: 4643-4652; Li et al., 2001, Proc Natl Acad Sci USA 98:4461-4465). As shown in Table 3, Pellino-1-mediated NF-kB activation isintact in mutant I1A and I3A cells, indicating that Pellino-1 functionsdownstream of IRAK. On the other hand, Pellino-1-induced NF-kBactivation was inhibited by a dominant-negative kinase-inactive TAK1mutant (TAK1 DN), indicating that Pellino-1 must function upstream ofTAK1 (Table 3). Taken together, the above results support the hypothesisthat the Pellino-1-IRAK-IRAK4-TRAF6 signaling complex functions betweenthe receptor complex (Complex I) and the TAK1 complex (Complex II).TABLE 3 In luceriferase reporter assays, overexpression of Pellino-1exerts an effect downstream of IRAK but upstream of TAK1 Fold Increasein Luceriferase Activity by Pellino-1 Transfection into Wild Type Amountof Pellino-1 and into IL-i-Unresponsive Mutant Recipient Cells DNAtransfected Wild Type (293 cells) I1A mutant I3A mutant 100 ng 1.1 3.21.1 250 ng 2.8 5.5 2.4 500 ng 5.9 5.4 4.5 Fold Increase in LuciferaseActivity Amount of TAK-1 DN by Pellino-1 Transfection in the DNAco-transfected Presence of Dominant-Negative TAK-1   0 ng 9.4  500 ng 81000 ng 5.4 3000 ng 1.4

[0214] We used siRNA, a new gene knock-down technology, to furtherinvestigate the functional role of Pellino-1 in IL-1-mediated signalingpathways. The discovery that potent sequence-specific inactivation ofgene function can be induced by double-stranded RNA (dsRNA) has led to arevolution in reverse genetic analysis. Synthetic 21-nucleotide RNAduplexes (siRNAs), base-paired so that they have two-nucleotide 3′overhangs, can specifically inhibit gene expression in cultured cells(Elbashir et al., 2001, Nature 411: 494-498). A mammalian expressionvector was developed that directs the synthesis of siRNA-liketranscripts (pSUPER, suppression of endogenous RNA, Brummelkamp et al.,2002, Science 296: 550-553). Vector pSUPER was obtained from Dr. ReuvenAgami's group (Center for Biomedical Genetics, Netherlands), and thePellino-1-silencing vector Pellino-1-pSUPER was generated according tothe methods of Brummelkamp et al. Forward and reverse primers weresynthesized, which contain 20 nucleotides from Pellino-1 (nucleotidesfrom position 47 to position 66 of the human Pellino-1 coding sequence,i.e. nucleotides 47 through 66 of SEQ ID NO:3) and a 9-base spacer,followed by the reverse complement of the same 20-nt sequence and clonedunder the H1-RNA gene promoter in the pSUPER vector. Pellino-1-pSUPERhas been stably transfected into 293 cells: 2×10⁵ 293 cells were seededonto a 10-cm plate and cotransfected the following day by the calciumphosphate method with 10 micrograms of the vector and 1 microgram ofpBabePuro which confers resistance to puromycin (Morgenstern and Land,1990, Nucleic Acids Res 18: 3587-3596). After 48 hours, the cells wereselected with 1 microgram/ml of puromycin until clones appeared. Clonesderived from this stable transfection were analyzed by Northernprocedure using Pellino-1 gene specific probe (FIG. 3, Panel A).Twenty-seven percent of the clones (clones C-1, C-3, C-12, C-16, andC-18) transfected with Pellino-1-pSUPER revealed 90% of reduction in thelevels of Pellino-1 mRNA as compared to the non-transfected cells (FIG.3, Panel A), whereas the clones transfected with vector pSUPER showedthe same levels of Pellino-1 mRNA (data not shown). It is clear thatRNAi produced from Pellino-1-pSUPER can indeed efficiently suppress theexpression of Pellino-1 mRNA. We then examined the effect of reducedexpression of Pellino-1 on an IL-1-mediated signaling pathway using anNF-kB gel-shift assay. An NF-kB binding site from the IP-10 gene wasused as a probe (the hIP-10 kB2 motif, as shown in FIG. 5A of Majumder,et al., 1998, J Neurosci. Res 54: 169-180). Complementaryoligonuclotides, end-labled with polynucleotide kinase (RocheDiagnostics, Indianapolis, Ind.) and gamma³²P-labeled ATP, were annealedby slow cooling. Approximately 20,000 cpm of probe were used per assay(Kessler et al., 1990, Genes Dev. 4: 1753-1765). Whole-cell extractswere used for the assay. The binding reaction was carried out at 4degrees C. for twenty minutes in a total volume of 20 microliterscontaining 20 mM Hepes buffer, pH 7.0, 10 mM KCl, 0.1% Nonidet P-40, 0.5mM dithiothreitol, 0.25 mM phenylmethanesulfonyl fluoride, and 10%glycerol. As shown in FIG. 3, Panel B, IL-1 induced much weaker NF-kBactivation in C-12 and C-16 (in which Pellino-1 expression is knockeddown, FIG. 3, Panel A) than in C-9 (a clone from the same transfectionwhose Pellino-1 expression is not altered, FIG. 3, Panel A), stronglysuggesting that Pellino-1 is required for IL-1-mediated NF-kBactivation. On the other hand, the reduced expression of Pellino-1 hadno effect on TNF-induced NF-kB activation, indicating that Pellino-1 isspecifically required for IL-1 signaling. We also examined the effect ofreduced Pellino-1 expression on IL-1-induced gene expression. As shownin FIG. 3, Panel C, IL-1-induced IL-8 gene expression was greatlyreduced in C-12 and C-16 as compared to that in C-9, confirming thatPellino-1 is indeed required for this IL-1-mediated signaling pathway.

[0215] We have identified Pellino-1 as a novel signaling molecule forthe IL-1 signaling pathway. We have shown that Pellino-1 is required forNF-kB activation and IL-8 gene expression, probably through itsinteraction with IRAK, IRAK4, and TRAF6. Our data also suggest that thePellino-1-IRAK-IRAK4-TRAF6 signaling complex is likely to be anintermediate, functioning between the receptor complex (Complex I) andthe TAK1 complex (Complex II). Release of the phosphorylated IRAK fromthe receptor is essential for mediating the downstream signaling eventsin response to IL-1 stimulation. However, it is unclear how thephosphorylated IRAK is released from the receptor. Pellino-1 forms acomplex with the phosphorylated IRAK but not with the IL-1 receptor,suggesting that Pellino-1 may play an important role in facilitating therelease of phosphorylated IRAK from the receptor. In addition, Pellino-1may participate in regulating the function of IRAK. The degradation ofthe modified IRAK is a delayed process, taking about six hours afterIL-1 stimulation. The fact that Pellino-1 interacts with the modifiedIRAK upon IL-1 stimulation (FIG. 2, Panel A) suggests that Pellino-1 mayplay an important role in preventing the modified IRAK from immediatedegradation.

[0216] The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan readily recognizes that many other embodiments areencompassed by the invention. The relevant disclosures of referencescited herein are specifically incorporated by reference.

[0217] Sequences Presented in the Sequence Listing SEQ ID NO SequenceType Description SEQ ID NO:1 Nucleotide Murine (Mus musculus) Pellino-1coding sequence SEQ ID NO:2 Amino acid Murine (Mus musculus) Pellino-1amino acid sequence SEQ ID NO:3 Nucleotide Human (Homo sapiens)Pellino-1 coding sequence SEQ ID NO:4 Amino acid Human (Homo sapiens)Pellino-1 amino acid sequence SEQ ID NO:5 Nucleotide Murine (Musmusculus) Pellino-2 coding sequence SEQ ID NO:6 Amino acid Murine (Musmusculus) Pellino-2 amino acid sequence SEQ ID NO:7 Nucleotide Human(Homo sapiens) Pellino-2 coding sequence SEQ ID NO:8 Amino acid Human(Homo sapiens) Pellino-2 amino acid sequence SEQ ID NO:9 Nucleotide PCRprimer SEQ ID NO:10 Nucleotide PCR primer SEQ ID NO:11 Nucleotide Human(Homo sapiens) Pellino-3 coding sequence SEQ ID NO:12 Amino acid Human(Homo sapiens) Pellino-3 amino acid sequence SEQ ID NO:13 Amino acidFruit fly (Drosophila melanogaster) Pellino (GenBank AAC96298) SEQ IDNO:14 Amino acid Ascidian (Sea squirt, Ciona intestinalis) Pellino(GenBank BAB00628) SEQ ID NO:15 Amino acid Nematode (Caenorhabditiselegans) Pellino (GenBank CAB97346)

[0218]

1 15 1 1257 DNA Mus musculus 1 atgttttctc ctgatcaaga aaatcatccttccaaagccc cagtaaaata tggcgaactc 60 attgtcttag gatataatgg atctctcccaaacggtgata gaggaaggag gaaaagtagg 120 tttgctttgt ttaaaagacc taaggcaaatggggtgaagc ctagcaccgt gcacattgca 180 tgtactcctc aggctgccaa ggcaataagcaacaaggacc agcatagcat atcatatact 240 ttatctcgag cccagacggt ggtggttgaatatactcatg acagcaacac tgatatgttt 300 cagattggtc ggtcaactga aagtcctattgattttgtag taactgacac cgttcctgga 360 agtcagagta attccgacac gcagtcagtacaaagcacta tatcaagatt tgcctgtagg 420 atcatatgtg agcgcagtcc cccctttacagctcggattt atgctgcagg gtttgattca 480 tcaaaaaaca tctttcttgg ggagaaggctgccaagtgga agacatctga tgggcagatg 540 gatggcttga ccactaatgg agttcttgtgatgcatccac gtaatgggtt cacagaagac 600 tccaaacctg gaatatggag agaaatatcagtatgtggga atgtcttcag tctgcgtgaa 660 accagatcag ctcagcagag aggaaagatggtggaaattg aaaccaatca gctacaagat 720 ggctccttaa ttgacctttg tggtgcaaccttgctgtggc gtactgcaga aggcctttcc 780 catactccta ctgtgaagca cttagaagctttaagacagg agatcaatgc agctcggccg 840 cagtgccctg tagggttcaa cacactagccttccccagta tgaagaggaa ggatgttgta 900 gatgaaaagc aaccatgggt atatctaaactgcggccatg tccatggtta tcataactgg 960 ggaaacaaag aagaacgtga cggcaaagatcgtgaatgtc ctatgtgtag gtctgttggt 1020 ccctatgtcc ctctgtggct tggatgtgaagctggatttt atgtggacgc cggccctccc 1080 acccatgcct ttagcccctg tgggcacgtgtgttcagaaa agacaacggc ttactggtcc 1140 cagatcccac ttcctcatgg tacgcacacttttcatgcag cctgcccctt ctgtgcacat 1200 cagttggctg gtgaacaagg ctatatcagacttattttcc aaggaccttt agactag 1257 2 418 PRT Mus musculus 2 Met Phe SerPro Asp Gln Glu Asn His Pro Ser Lys Ala Pro Val Lys 1 5 10 15 Tyr GlyGlu Leu Ile Val Leu Gly Tyr Asn Gly Ser Leu Pro Asn Gly 20 25 30 Asp ArgGly Arg Arg Lys Ser Arg Phe Ala Leu Phe Lys Arg Pro Lys 35 40 45 Ala AsnGly Val Lys Pro Ser Thr Val His Ile Ala Cys Thr Pro Gln 50 55 60 Ala AlaLys Ala Ile Ser Asn Lys Asp Gln His Ser Ile Ser Tyr Thr 65 70 75 80 LeuSer Arg Ala Gln Thr Val Val Val Glu Tyr Thr His Asp Ser Asn 85 90 95 ThrAsp Met Phe Gln Ile Gly Arg Ser Thr Glu Ser Pro Ile Asp Phe 100 105 110Val Val Thr Asp Thr Val Pro Gly Ser Gln Ser Asn Ser Asp Thr Gln 115 120125 Ser Val Gln Ser Thr Ile Ser Arg Phe Ala Cys Arg Ile Ile Cys Glu 130135 140 Arg Ser Pro Pro Phe Thr Ala Arg Ile Tyr Ala Ala Gly Phe Asp Ser145 150 155 160 Ser Lys Asn Ile Phe Leu Gly Glu Lys Ala Ala Lys Trp LysThr Ser 165 170 175 Asp Gly Gln Met Asp Gly Leu Thr Thr Asn Gly Val LeuVal Met His 180 185 190 Pro Arg Asn Gly Phe Thr Glu Asp Ser Lys Pro GlyIle Trp Arg Glu 195 200 205 Ile Ser Val Cys Gly Asn Val Phe Ser Leu ArgGlu Thr Arg Ser Ala 210 215 220 Gln Gln Arg Gly Lys Met Val Glu Ile GluThr Asn Gln Leu Gln Asp 225 230 235 240 Gly Ser Leu Ile Asp Leu Cys GlyAla Thr Leu Leu Trp Arg Thr Ala 245 250 255 Glu Gly Leu Ser His Thr ProThr Val Lys His Leu Glu Ala Leu Arg 260 265 270 Gln Glu Ile Asn Ala AlaArg Pro Gln Cys Pro Val Gly Phe Asn Thr 275 280 285 Leu Ala Phe Pro SerMet Lys Arg Lys Asp Val Val Asp Glu Lys Gln 290 295 300 Pro Trp Val TyrLeu Asn Cys Gly His Val His Gly Tyr His Asn Trp 305 310 315 320 Gly AsnLys Glu Glu Arg Asp Gly Lys Asp Arg Glu Cys Pro Met Cys 325 330 335 ArgSer Val Gly Pro Tyr Val Pro Leu Trp Leu Gly Cys Glu Ala Gly 340 345 350Phe Tyr Val Asp Ala Gly Pro Pro Thr His Ala Phe Ser Pro Cys Gly 355 360365 His Val Cys Ser Glu Lys Thr Thr Ala Tyr Trp Ser Gln Ile Pro Leu 370375 380 Pro His Gly Thr His Thr Phe His Ala Ala Cys Pro Phe Cys Ala His385 390 395 400 Gln Leu Ala Gly Glu Gln Gly Tyr Ile Arg Leu Ile Phe GlnGly Pro 405 410 415 Leu Asp 3 1257 DNA Homo sapiens 3 atgttttctcctgatcaaga aaatcatcca tctaaagcac cagtaaaata tggtgaactc 60 attgtcttaggatataatgg atctctccca aacggtgata gaggaaggag gaaaagtagg 120 tttgctttgtttaaaagacc taaggcaaat ggggtgaagc ccagcactgt gcatattgct 180 tgtactcctcaggctgcaaa ggcaataagc aacaaagacc agcatagcat atcatatact 240 ttatctcgggcccagactgt ggtggttgaa tatactcatg acagcaacac cgatatgttt 300 cagattggccggtcgactga aagccccatt gattttgtag taactgacac ggttcctgga 360 agtcaaagtaattctgatac acagtcagta caaagcacta tatcaagatt tgcctgcaga 420 atcatatgtgaacggaatcc tccctttaca gcacggattt atgctgcagg gtttgactca 480 tcaaaaaacatctttcttgg ggagaaggct gccaaatgga agacatcaga tggacagatg 540 gatggcttgaccactaatgg tgttcttgtg atgcatccac gcaatgggtt cacagaagac 600 tccaagcctggaatatggag agaaatatcg gtgtgtggga atgtatttag cctacgtgaa 660 accagatcggctcagcagag aggaaaaatg gtggaaattg aaaccaatca gttacaagat 720 ggctcgttaattgacctctg tggtgcaaca ttgttatggc gtactgcaga aggcctttcc 780 cacactcctaccgtgaagca tttagaagct ttaagacagg aaatcaatgc agcacgacct 840 cagtgccctgtagggttcaa cacactagca tttcctagta tgaagaggaa agacgttgta 900 gatgaaaaacaaccatgggt atatctaaac tgcggccatg tacatggcta tcataactgg 960 ggaaacaaagaagaacgtga tggcaaagat cgtgaatgtc ctatgtgtag gtctgttggt 1020 ccctatgttcctctgtggct tggatgtgaa gctggatttt atgtggacgc cggccctcca 1080 acccatgcgtttagcccgtg tgggcatgtg tgttcagaaa agacaactgc ctattggtcc 1140 cagatcccacttcctcatgg tactcatact tttcatgcag cctgtccctt ttgtgcacat 1200 cagttggctggtgaacaagg ctacatcaga cttatttttc aaggacctct agactaa 1257 4 418 PRT Homosapiens 4 Met Phe Ser Pro Asp Gln Glu Asn His Pro Ser Lys Ala Pro ValLys 1 5 10 15 Tyr Gly Glu Leu Ile Val Leu Gly Tyr Asn Gly Ser Leu ProAsn Gly 20 25 30 Asp Arg Gly Arg Arg Lys Ser Arg Phe Ala Leu Phe Lys ArgPro Lys 35 40 45 Ala Asn Gly Val Lys Pro Ser Thr Val His Ile Ala Cys ThrPro Gln 50 55 60 Ala Ala Lys Ala Ile Ser Asn Lys Asp Gln His Ser Ile SerTyr Thr 65 70 75 80 Leu Ser Arg Ala Gln Thr Val Val Val Glu Tyr Thr HisAsp Ser Asn 85 90 95 Thr Asp Met Phe Gln Ile Gly Arg Ser Thr Glu Ser ProIle Asp Phe 100 105 110 Val Val Thr Asp Thr Val Pro Gly Ser Gln Ser AsnSer Asp Thr Gln 115 120 125 Ser Val Gln Ser Thr Ile Ser Arg Phe Ala CysArg Ile Ile Cys Glu 130 135 140 Arg Asn Pro Pro Phe Thr Ala Arg Ile TyrAla Ala Gly Phe Asp Ser 145 150 155 160 Ser Lys Asn Ile Phe Leu Gly GluLys Ala Ala Lys Trp Lys Thr Ser 165 170 175 Asp Gly Gln Met Asp Gly LeuThr Thr Asn Gly Val Leu Val Met His 180 185 190 Pro Arg Asn Gly Phe ThrGlu Asp Ser Lys Pro Gly Ile Trp Arg Glu 195 200 205 Ile Ser Val Cys GlyAsn Val Phe Ser Leu Arg Glu Thr Arg Ser Ala 210 215 220 Gln Gln Arg GlyLys Met Val Glu Ile Glu Thr Asn Gln Leu Gln Asp 225 230 235 240 Gly SerLeu Ile Asp Leu Cys Gly Ala Thr Leu Leu Trp Arg Thr Ala 245 250 255 GluGly Leu Ser His Thr Pro Thr Val Lys His Leu Glu Ala Leu Arg 260 265 270Gln Glu Ile Asn Ala Ala Arg Pro Gln Cys Pro Val Gly Phe Asn Thr 275 280285 Leu Ala Phe Pro Ser Met Lys Arg Lys Asp Val Val Asp Glu Lys Gln 290295 300 Pro Trp Val Tyr Leu Asn Cys Gly His Val His Gly Tyr His Asn Trp305 310 315 320 Gly Asn Lys Glu Glu Arg Asp Gly Lys Asp Arg Glu Cys ProMet Cys 325 330 335 Arg Ser Val Gly Pro Tyr Val Pro Leu Trp Leu Gly CysGlu Ala Gly 340 345 350 Phe Tyr Val Asp Ala Gly Pro Pro Thr His Ala PheSer Pro Cys Gly 355 360 365 His Val Cys Ser Glu Lys Thr Thr Ala Tyr TrpSer Gln Ile Pro Leu 370 375 380 Pro His Gly Thr His Thr Phe His Ala AlaCys Pro Phe Cys Ala His 385 390 395 400 Gln Leu Ala Gly Glu Gln Gly TyrIle Arg Leu Ile Phe Gln Gly Pro 405 410 415 Leu Asp 5 1260 DNA Musmusculus 5 atgttttccc cgggccagga ggaacccagc gcccccaaca aggagccggtgaaatacggg 60 gagctggtgg tcctggggta caatggtgct ttacctaatg gtgacaggggcaggaggaaa 120 agcagatttg ccctctataa gcggacctac gccagtggtg tcaaacccagcacaatccac 180 atggtctcca caccacaggc gtccaaggcc atcagctcca gaggacatcacagcatatcg 240 tacacgttgt cacggagcca gacggtagtg gtggagtaca cacacgataaagacacggac 300 atgtttcagg tgggcaggtc aacagaaagc cccattgact tcgtggtcacagacacggtt 360 tccggcggtc agaacgaaga tgcccagatc acacagagca ccatctctaggttcgcatgc 420 aggatcgtgt gtgacaggaa cgagccatat acagcacgca tattcgcggcaggattcgat 480 tcttccaaaa atatctttct tggagagaaa gcagcaaaat ggaaaaaccctgatggacac 540 atggatggac tcactaccaa tggtgtccta gtgatgcacc cgcaaggaggcttcaccgag 600 gaatcccagc ctggagtctg gagagagatc tctgtctgtg gggatgtgtacaccttgcga 660 gagaccaggt cggcccagca gaggggaaag ctggtggaaa gtgagaccaacgtcctgcaa 720 gacggctccc tcattgacct gtgtggggcc actctcctct ggagaaccgcagatggcctt 780 tttcacgctc ctactcagaa gcacatagaa gccctccggc aggagatcaatgcagcccga 840 ccccagtgcc ccgtgggcct taacaccctg gccttcccca gcatcaaccggaaggaagtg 900 gtggaagaga agcagccctg ggcatacctg agctgcggcc atgtgcacggctaccacagc 960 tggggccatc ggagcgacgc ggaagccaac gagagggagt gtcccatgtgcaggactgtg 1020 ggcccctacg tccctctctg gctgggctgt gaggcaggat tttatgtcgatgcgggaccc 1080 ccaactcacg ctttcacccc ctgcgggcac gtctgttcag aaaagtctgccaagtactgg 1140 tcgcagatcc cactgcccca cggaacgcac gcgtttcatg ccgcctgtccgttctgcgcc 1200 acgcagctgg ttggtgaaca gaactgcatc aaattgattt tccaaggtccagtggactga 1260 6 419 PRT Mus musculus 6 Met Phe Ser Pro Gly Gln Glu GluPro Ser Ala Pro Asn Lys Glu Pro 1 5 10 15 Val Lys Tyr Gly Glu Leu ValVal Leu Gly Tyr Asn Gly Ala Leu Pro 20 25 30 Asn Gly Asp Arg Gly Arg ArgLys Ser Arg Phe Ala Leu Tyr Lys Arg 35 40 45 Thr Tyr Ala Ser Gly Val LysPro Ser Thr Ile His Met Val Ser Thr 50 55 60 Pro Gln Ala Ser Lys Ala IleSer Ser Arg Gly His His Ser Ile Ser 65 70 75 80 Tyr Thr Leu Ser Arg SerGln Thr Val Val Val Glu Tyr Thr His Asp 85 90 95 Lys Asp Thr Asp Met PheGln Val Gly Arg Ser Thr Glu Ser Pro Ile 100 105 110 Asp Phe Val Val ThrAsp Thr Val Ser Gly Gly Gln Asn Glu Asp Ala 115 120 125 Gln Ile Thr GlnSer Thr Ile Ser Arg Phe Ala Cys Arg Ile Val Cys 130 135 140 Asp Arg AsnGlu Pro Tyr Thr Ala Arg Ile Phe Ala Ala Gly Phe Asp 145 150 155 160 SerSer Lys Asn Ile Phe Leu Gly Glu Lys Ala Ala Lys Trp Lys Asn 165 170 175Pro Asp Gly His Met Asp Gly Leu Thr Thr Asn Gly Val Leu Val Met 180 185190 His Pro Gln Gly Gly Phe Thr Glu Glu Ser Gln Pro Gly Val Trp Arg 195200 205 Glu Ile Ser Val Cys Gly Asp Val Tyr Thr Leu Arg Glu Thr Arg Ser210 215 220 Ala Gln Gln Arg Gly Lys Leu Val Glu Ser Glu Thr Asn Val LeuGln 225 230 235 240 Asp Gly Ser Leu Ile Asp Leu Cys Gly Ala Thr Leu LeuTrp Arg Thr 245 250 255 Ala Asp Gly Leu Phe His Ala Pro Thr Gln Lys HisIle Glu Ala Leu 260 265 270 Arg Gln Glu Ile Asn Ala Ala Arg Pro Gln CysPro Val Gly Leu Asn 275 280 285 Thr Leu Ala Phe Pro Ser Ile Asn Arg LysGlu Val Val Glu Glu Lys 290 295 300 Gln Pro Trp Ala Tyr Leu Ser Cys GlyHis Val His Gly Tyr His Ser 305 310 315 320 Trp Gly His Arg Ser Asp AlaGlu Ala Asn Glu Arg Glu Cys Pro Met 325 330 335 Cys Arg Thr Val Gly ProTyr Val Pro Leu Trp Leu Gly Cys Glu Ala 340 345 350 Gly Phe Tyr Val AspAla Gly Pro Pro Thr His Ala Phe Thr Pro Cys 355 360 365 Gly His Val CysSer Glu Lys Ser Ala Lys Tyr Trp Ser Gln Ile Pro 370 375 380 Leu Pro HisGly Thr His Ala Phe His Ala Ala Cys Pro Phe Cys Ala 385 390 395 400 ThrGln Leu Val Gly Glu Gln Asn Cys Ile Lys Leu Ile Phe Gln Gly 405 410 415Pro Val Asp 7 1263 DNA Homo sapiens 7 atgttttccc ctggccagga ggaacactgcgcccccaata aggagccagt gaaatacggg 60 gagctggtgg tgctcgggta caatggtgctttacccaatg gagatagagg acggaggaaa 120 agtagatttg ccctctacaa gcggcccaaggcaaatggtg tcaaacccag caccgtccat 180 gtgatatcca cgccccaggc atccaaggctatcagctgca aaggtcaaca cagtatatcc 240 tacactttgt caaggaatca gactgtggtggtggagtaca cacatgataa ggatacggat 300 atgtttcagg tgggcagatc aacagaaagccctatcgact tcgttgtcac agacacgatt 360 tctggcagcc agaacacgga cgaagcccagatcacacaga gcaccatatc caggttcgcc 420 tgcaggatcg tgtgcgacag gaatgaaccttacacagcac ggatattcgc cgccggattt 480 gactcttcca aaaacatatt tcttggagtaaaggcagcaa agtggaaaaa ccccgacggc 540 cacatggatg ggctcactac taatggcgtcctggtgatgc atccacgagg gggcttcacc 600 gaggagtccc agcccggggt ctggcgcgagatctctgtct gtggagatgt gtacaccttg 660 cgagaaacca ggtcggccca gcaacgaggaaagctggtgg aaagtgagac caacgtcctg 720 caggacggct ccctcattga cctgtgtggggccactctcc tctggagaac agcagatggg 780 ctttttcata ctccaactca gaagcacatagaagccctcc ggcaggagat taacgccgcc 840 cggcctcagt gtcctgtggg gctcaacaccctggccttcc ccagcatcaa caggaaagag 900 gtggtggagg agaagcagcc ctgggcatatctcagttgtg gccacgtgca cgggtaccac 960 aactggggcc atcggagtga cacggaggccaacgagaggg agtgtcccat gtgcaggact 1020 gtgggcccct atgtgcctct ctggcttggctgtgaggcag gattttatgt agacgcagga 1080 ccgccaactc atgctttcac tccctgtggacacgtgtgct cggagaagtc tgcaaaatac 1140 tggtctcaga tcccgttgcc tcatggaactcatgcatttc acgctgcttg ccctttctgt 1200 gctacacagc tggttgggga gcaaaactgcatcaaattaa ttttccaagg tccaattgac 1260 tga 1263 8 420 PRT Homo sapiens 8Met Phe Ser Pro Gly Gln Glu Glu His Cys Ala Pro Asn Lys Glu Pro 1 5 1015 Val Lys Tyr Gly Glu Leu Val Val Leu Gly Tyr Asn Gly Ala Leu Pro 20 2530 Asn Gly Asp Arg Gly Arg Arg Lys Ser Arg Phe Ala Leu Tyr Lys Arg 35 4045 Pro Lys Ala Asn Gly Val Lys Pro Ser Thr Val His Val Ile Ser Thr 50 5560 Pro Gln Ala Ser Lys Ala Ile Ser Cys Lys Gly Gln His Ser Ile Ser 65 7075 80 Tyr Thr Leu Ser Arg Asn Gln Thr Val Val Val Glu Tyr Thr His Asp 8590 95 Lys Asp Thr Asp Met Phe Gln Val Gly Arg Ser Thr Glu Ser Pro Ile100 105 110 Asp Phe Val Val Thr Asp Thr Ile Ser Gly Ser Gln Asn Thr AspGlu 115 120 125 Ala Gln Ile Thr Gln Ser Thr Ile Ser Arg Phe Ala Cys ArgIle Val 130 135 140 Cys Asp Arg Asn Glu Pro Tyr Thr Ala Arg Ile Phe AlaAla Gly Phe 145 150 155 160 Asp Ser Ser Lys Asn Ile Phe Leu Gly Val LysAla Ala Lys Trp Lys 165 170 175 Asn Pro Asp Gly His Met Asp Gly Leu ThrThr Asn Gly Val Leu Val 180 185 190 Met His Pro Arg Gly Gly Phe Thr GluGlu Ser Gln Pro Gly Val Trp 195 200 205 Arg Glu Ile Ser Val Cys Gly AspVal Tyr Thr Leu Arg Glu Thr Arg 210 215 220 Ser Ala Gln Gln Arg Gly LysLeu Val Glu Ser Glu Thr Asn Val Leu 225 230 235 240 Gln Asp Gly Ser LeuIle Asp Leu Cys Gly Ala Thr Leu Leu Trp Arg 245 250 255 Thr Ala Asp GlyLeu Phe His Thr Pro Thr Gln Lys His Ile Glu Ala 260 265 270 Leu Arg GlnGlu Ile Asn Ala Ala Arg Pro Gln Cys Pro Val Gly Leu 275 280 285 Asn ThrLeu Ala Phe Pro Ser Ile Asn Arg Lys Glu Val Val Glu Glu 290 295 300 LysGln Pro Trp Ala Tyr Leu Ser Cys Gly His Val His Gly Tyr His 305 310 315320 Asn Trp Gly His Arg Ser Asp Thr Glu Ala Asn Glu Arg Glu Cys Pro 325330 335 Met Cys Arg Thr Val Gly Pro Tyr Val Pro Leu Trp Leu Gly Cys Glu340 345 350 Ala Gly Phe Tyr Val Asp Ala Gly Pro Pro Thr His Ala Phe ThrPro 355 360 365 Cys Gly His Val Cys Ser Glu Lys Ser Ala Lys Tyr Trp SerGln Ile 370 375 380 Pro Leu Pro His Gly Thr His Ala Phe His Ala Ala CysPro Phe Cys 385 390 395 400 Ala Thr Gln Leu Val Gly Glu Gln Asn Cys IleLys Leu Ile Phe Gln 405 410 415 Gly Pro Ile Asp 420 9 35 DNA ArtificialSequence olignonucleotide primer 9 atattcactg aattctgatg ttttctcctgatcaa 35 10 37 DNA Artificial Sequence oligonucleotide primer 10agtgaatatg aattcctact tatcatcgtc atctttg 37 11 1338 DNA Homo sapiensmisc_feature (513)..(513) unsure 11 atggtgctgg aaggaaaccc tgaagtggggtccccccgaa cctcagacct ccagcaccgg 60 gggaacaagg gctcttgcgt tctctcctctcccggtgaag atgcgcagcc aggcgaggag 120 cccatcaagt atggtgaact catcgtcctgggctacaatg gttgtctggc aagtggggac 180 aagggccgcc ggcgaagccg cctggcactgagccgccggt cgcacgccaa cggggtgaag 240 ccagacgtca tgcaccacat ctccacgccgctcgtctcca aggcactgag taaccgtggt 300 cagcacagca tctcgtatac actgtcccggagccactcgg tcatagtgga gtatacacat 360 gatagcgaca cagacatgtt ccagattggccgctccacag agaacatgat tgacttcgtg 420 gtaacagaca cgtcccctgg aggaggggctgccgagggcc cttctgccca gagcaccatc 480 tcccgctatg cctgccgcat cctctgtgaccgncggccac cctatactgc ccgcatctat 540 gccgctggct tcgatgcctc tagcaacatcttccttggag agcgagcggc caaatggcgg 600 accccagatg gcctgatgga tggactgaccaccaatggag tcctggtgat gcacccggca 660 ggcggcttct ccgaggactc agccccgggtgtctggcggg agatctcggt ctgtgggaat 720 gtgtacacat tgcgggacag ccgctcagcccagcagcggg gcaagctggt agaaaacgag 780 tccaacgtgc tgcaggacgg ctctctcatcgacctgtgtg gggccacact gctgtggcgc 840 acaccggcgg ggctgctgcg ggctcccacactgaagcaac tggaggccca gcggcaggag 900 gcaaatgcag cgcgccccca gtgccccgtgggcctcagca ctctggcctt ccccagccca 960 gcccgtggcc gcacagcgcc cgacaaacagcagccctggg tctacgtccg ctgcgggcac 1020 gtccatggct accacggctg gggctgccggcgggagcggg gcccccagga gcgcgaatgt 1080 cctctctgcc gccttgtggg gccttatgtgcctctatggc ttggccagga ggccggcctc 1140 tgcctggacc ctgggccgcc tagccatgcctttgcacctt gcggccacgt ctgctctgag 1200 aagactgccc gctactgggc ccagacaccactgccccacg gcacccatgc tttccatgcc 1260 gcctgcccct tttgcggggc ctggcttaccggcgagcatg gctgcgtccg cctcattttc 1320 cagggcccgc tggattag 1338 12 445PRT Homo sapiens 12 Met Val Leu Glu Gly Asn Pro Glu Val Gly Ser Pro ArgThr Ser Asp 1 5 10 15 Leu Gln His Arg Gly Asn Lys Gly Ser Cys Val LeuSer Ser Pro Gly 20 25 30 Glu Asp Ala Gln Pro Gly Glu Glu Pro Ile Lys TyrGly Glu Leu Ile 35 40 45 Val Leu Gly Tyr Asn Gly Cys Leu Ala Ser Gly AspLys Gly Arg Arg 50 55 60 Arg Ser Arg Leu Ala Leu Ser Arg Arg Ser His AlaAsn Gly Val Lys 65 70 75 80 Pro Asp Val Met His His Ile Ser Thr Pro LeuVal Ser Lys Ala Leu 85 90 95 Ser Asn Arg Gly Gln His Ser Ile Ser Tyr ThrLeu Ser Arg Ser His 100 105 110 Ser Val Ile Val Glu Tyr Thr His Asp SerAsp Thr Asp Met Phe Gln 115 120 125 Ile Gly Arg Ser Thr Glu Asn Met IleAsp Phe Val Val Thr Asp Thr 130 135 140 Ser Pro Gly Gly Gly Ala Ala GluGly Pro Ser Ala Gln Ser Thr Ile 145 150 155 160 Ser Arg Tyr Ala Cys ArgIle Leu Cys Asp Arg Arg Pro Pro Tyr Thr 165 170 175 Ala Arg Ile Tyr AlaAla Gly Phe Asp Ala Ser Ser Asn Ile Phe Leu 180 185 190 Gly Glu Arg AlaAla Lys Trp Arg Thr Pro Asp Gly Leu Met Asp Gly 195 200 205 Leu Thr ThrAsn Gly Val Leu Val Met His Pro Ala Gly Gly Phe Ser 210 215 220 Glu AspSer Ala Pro Gly Val Trp Arg Glu Ile Ser Val Cys Gly Asn 225 230 235 240Val Tyr Thr Leu Arg Asp Ser Arg Ser Ala Gln Gln Arg Gly Lys Leu 245 250255 Val Glu Asn Glu Ser Asn Val Leu Gln Asp Gly Ser Leu Ile Asp Leu 260265 270 Cys Gly Ala Thr Leu Leu Trp Arg Thr Pro Ala Gly Leu Leu Arg Ala275 280 285 Pro Thr Leu Lys Gln Leu Glu Ala Gln Arg Gln Glu Ala Asn AlaAla 290 295 300 Arg Pro Gln Cys Pro Val Gly Leu Ser Thr Leu Ala Phe ProSer Pro 305 310 315 320 Ala Arg Gly Arg Thr Ala Pro Asp Lys Gln Gln ProTrp Val Tyr Val 325 330 335 Arg Cys Gly His Val His Gly Tyr His Gly TrpGly Cys Arg Arg Glu 340 345 350 Arg Gly Pro Gln Glu Arg Glu Cys Pro LeuCys Arg Leu Val Gly Pro 355 360 365 Tyr Val Pro Leu Trp Leu Gly Gln GluAla Gly Leu Cys Leu Asp Pro 370 375 380 Gly Pro Pro Ser His Ala Phe AlaPro Cys Gly His Val Cys Ser Glu 385 390 395 400 Lys Thr Ala Arg Tyr TrpAla Gln Thr Pro Leu Pro His Gly Thr His 405 410 415 Ala Phe His Ala AlaCys Pro Phe Cys Gly Ala Trp Leu Thr Gly Glu 420 425 430 His Gly Cys ValArg Leu Ile Phe Gln Gly Pro Leu Asp 435 440 445 13 424 PRT Drosophilamelanogaster 13 Met Val Lys Arg Thr Asp Gly Thr Glu Ser Pro Ile Leu AlaGlu Asp 1 5 10 15 Gly Gly Asp Gly His Asp Lys Pro Arg Leu Arg Tyr GlyGlu Leu Val 20 25 30 Ile Leu Gly Tyr Asn Gly Tyr Leu Pro Gln Gly Asp ArgGly Arg Arg 35 40 45 Arg Ser Lys Phe Val Leu His Lys Arg Thr Glu Ala SerGly Val Lys 50 55 60 Arg Ser Lys His Tyr Ile Val Gln Ser Pro Gln Thr SerLys Ala Ile 65 70 75 80 Leu Asp Ala Asn Gln His Ser Ile Ser Tyr Thr LeuSer Arg Asn Gln 85 90 95 Ala Val Ile Val Glu Tyr Lys Glu Asp Thr Glu ThrAsp Met Phe Gln 100 105 110 Val Gly Arg Ser Ser Glu Ser Pro Ile Asp PheVal Val Met Asp Thr 115 120 125 Leu Pro Gly Asp Lys Lys Asp Ala Lys ValMet Gln Ser Thr Ile Ser 130 135 140 Arg Phe Ala Cys Arg Ile Leu Val AsnArg Cys Glu Pro Ala Lys Ala 145 150 155 160 Arg Ile Phe Ala Ala Gly PheAsp Ser Ser Arg Asn Ile Phe Leu Gly 165 170 175 Glu Lys Ala Thr Lys TrpGln Asp Asn Val Glu Ile Asp Gly Leu Thr 180 185 190 Thr Asn Gly Val LeuIle Met His Pro Lys Gly Ser Phe Cys Gly Gly 195 200 205 Asn Ala Lys CysGly Leu Trp Arg Glu Cys Ser Val Gly Gly Asp Val 210 215 220 Phe Ser LeuArg Glu Ser Arg Ser Ala Gln Gln Lys Gly Gln Pro Ile 225 230 235 240 TyrAsp Glu Cys Asn Ile Leu Gln Asp Gly Thr Leu Ile Asp Leu Cys 245 250 255Gly Ala Thr Leu Leu Trp Arg Ser Ala Glu Gly Leu Gln His Ser Pro 260 265270 Thr Lys His Asp Leu Glu Lys Leu Ile Asp Ala Ile Asn Ala Gly Arg 275280 285 Pro Gln Cys Pro Val Gly Leu Asn Thr Leu Val Ile Pro Arg Lys Val290 295 300 Asn Ile Gly Asp Gln Val Asn Gln Pro Tyr Val Tyr Leu Asn CysGly 305 310 315 320 His Val Gln Gly His His Asp Trp Gly Gln Asp Glu AsnThr Gly Ala 325 330 335 Arg Arg Cys Pro Met Cys Leu Glu Leu Gly Pro ValVal Thr Leu Cys 340 345 350 Met Gly Leu Glu Pro Ala Phe Tyr Val Asp ValGly Ala Pro Thr Tyr 355 360 365 Ala Phe Asn Pro Cys Gly His Met Ala ThrGlu Lys Thr Val Lys Tyr 370 375 380 Trp Ala Asn Val Glu Ile Pro His GlyThr Asn Gly Phe Gln Ala Val 385 390 395 400 Cys Pro Phe Cys Ala Thr ProLeu Asp Gly Ala Thr Gly Tyr Ile Lys 405 410 415 Leu Ile Phe Gln Asp AsnLeu Asp 420 14 455 PRT Ciona intestinalis 14 Met Lys Gln Glu Gly Met AspVal Ser Ala Ser Pro Ala Leu Ala Val 1 5 10 15 Ala Gly Gly Met Pro MetAsp Ile Gln Phe Glu Ala Gly Ala Ser Tyr 20 25 30 His Asn Phe Ser Gln GluAsp Ala Pro Lys Glu Asp Glu Gly Asp Ile 35 40 45 Ile Tyr Gly Gln Leu IleVal Leu Gly Thr Asn Gly Gln Leu Pro Thr 50 55 60 Gly Asp Lys Gly Arg ArgArg Ser Cys Phe Thr Leu Arg Arg Lys Arg 65 70 75 80 Lys Ala Thr Gly ValLys Pro Ser Asp Gln His Gln Val Tyr Gln Lys 85 90 95 Ala Ser His Ser GluThr Phe Leu Ser Lys Asp His His Ser Val Ser 100 105 110 Tyr Thr Leu ProArg Ser Val Val Val Val Pro Tyr Val His Asp Asp 115 120 125 Asn Ser AspMet Phe Gln Ile Gly Arg Ser Thr Glu Glu Pro Ile Asp 130 135 140 Phe ValLeu Met Asp Ile Glu Ala Gly Ser Ser Ile Pro Thr Asn His 145 150 155 160Lys Pro Gln Thr Gln Pro Lys Gln Ser Thr Ile Ser Arg Phe Ala Cys 165 170175 Arg Ile Val Cys Asp Arg Glu His Pro Tyr Thr Ser Arg Ile Tyr Ala 180185 190 Ala Gly Phe Asp Thr Ser Met Asn Ile Ile Leu Gly Glu Lys Ala Pro195 200 205 Lys Trp Thr Thr Glu Gln Asn Gly Lys Lys Ile Ile Asp Gly LeuThr 210 215 220 Thr Asn Gly Val Leu Ile Met Gln Pro Lys Asn Gly Phe SerGlu Ser 225 230 235 240 Ser Thr Pro Thr Gln Trp Lys Glu Thr Ser Val CysGly Asn Ile Tyr 245 250 255 Gln Leu Arg Glu Ser Arg Ser Ala Gln Leu ProGly Ile Arg Met Pro 260 265 270 Glu Asp Asn Asn Val Leu Val Asn Gly ThrLeu Ile Asp Leu Cys Gly 275 280 285 Ala Thr Leu Leu Trp Arg Ser Ser SerHis Glu Arg Cys Met Pro Thr 290 295 300 Pro Leu His Ile Asp Glu Leu IleHis Lys Leu Asn Leu Gly Arg Pro 305 310 315 320 Gln Cys Pro Val Gly LeuThr Thr Leu Ala Phe Pro Arg Arg Ser Lys 325 330 335 Ala Thr Lys Glu ThrGlu Lys Gln Pro Trp Val Tyr Leu Gln Cys Gly 340 345 350 His Val His GlyArg Ile Glu Trp Gly Tyr Gln Gly Glu Glu Glu Arg 355 360 365 Ile Cys ProLeu Cys Arg Ser Val Gly Lys Tyr Val Pro Leu Trp Val 370 375 380 Gly GlyGlu Pro Ala Phe Tyr Val Asp Ile Gly Pro Pro Ser Tyr Cys 385 390 395 400Phe Val Pro Cys Gly His Val Cys Ser Gln Lys Thr Ala Ile Tyr Trp 405 410415 Ser Gln Thr Ala Leu Pro His Gly Thr Gln Ala Tyr Ser Ala Ala Cys 420425 430 Pro Phe Cys Ala Thr Pro Leu Glu Gly Asp Leu Gly Tyr Lys Lys Leu435 440 445 Ile Phe Gln Gln Pro Leu Asp 450 455 15 458 PRTCaenorhabditis elegans 15 Met Val Asp Glu Ser Glu Leu Glu Asn Gly ThrPro Ser Pro Pro Ala 1 5 10 15 Tyr Ser Asn Glu Ala Ile Leu Asp Asp AspIle Tyr Gly Glu Leu Ile 20 25 30 Leu Leu Gly Phe Asn Gly Gln Ala Glu AsnArg Ala Thr Ser Lys Arg 35 40 45 Tyr Leu Thr Glu Lys Val Leu Arg Arg ArgAsp Ser Ala Asn Gly Ile 50 55 60 Lys Lys Cys Thr Val His Asn Val Ser ThrSer Asp Thr Lys Leu Thr 65 70 75 80 Lys Asp Lys Ala Arg His Thr Val SerPhe His Ser Asp Ser Asn Lys 85 90 95 Ser Val Val Ile Glu Tyr Ala Ala AspPro Ser Lys Asp Met Phe Gln 100 105 110 Ile Gly Arg Ala Ser Asp Asp GlnIle Asp Phe Thr Val Ile Asp Thr 115 120 125 Trp Met Phe Leu Pro Glu HisSer Asp Ala Ala Val Pro Ala Arg Pro 130 135 140 Gln Ile Asp Val Leu GluLys Gly Asp Arg Thr Ser Thr Ile Ser Arg 145 150 155 160 Phe Ala Cys ArgIle Leu Ile Asp Arg Glu Asn Ser Asn Lys Ala Tyr 165 170 175 Leu Tyr AlaAla Gly Phe Asp Ala His Gln Asn Ile Ser Ile Asn Lys 180 185 190 Lys SerLeu Lys Trp Thr Lys Ser Asn Gly Glu Val Asp Gly Leu Thr 195 200 205 ThrAsn Gly Val Leu Leu Leu His Pro Asn Lys Asp Asp Leu Leu Asp 210 215 220Asp Thr Val Asp Lys Pro Met Tyr Lys Trp Arg Glu Val Ser Ile Asn 225 230235 240 Gly Asp Val Tyr Glu Pro Arg Val Thr Arg Ser Ser Ser Ala Lys Gly245 250 255 Val Phe Val Pro Glu Trp Thr Asn Met Leu Gln Asp Gly Thr LeuIle 260 265 270 Asp Leu Cys Gly Ala Thr Ile Leu Trp Arg Thr Ala Asp GlyLeu Glu 275 280 285 Arg Ser Pro Lys Met Arg Glu Leu Glu Met Ala Leu AspArg Leu Ser 290 295 300 Ala Gly Arg Pro Gln Cys Pro Val Asn Leu Asn ThrLeu Val Ile Pro 305 310 315 320 Lys Lys Arg Asn Gly Arg Gln Ile Asn ArgArg Gln Pro Tyr Val Tyr 325 330 335 Leu Gln Cys Gly His Val Gln Gly ArgHis Glu Trp Gly Val Gln Glu 340 345 350 Asn Ser Gly Gln Arg Ser Gly LysCys Pro Ile Cys Leu Val Glu Ser 355 360 365 Glu Arg Ile Val Gln Leu SerMet Gly Met Glu Pro Ser Phe His Leu 370 375 380 Asp Ser Gly Val Leu AspHis Thr Phe Asn Pro Cys Gly His Met Ala 385 390 395 400 Ser Lys Gln ThrVal Leu Tyr Trp Ser Arg Ile Pro Leu Pro Gln Gly 405 410 415 Thr Cys ArgTyr Asp Pro Val Cys Pro Phe Cys Tyr Gln Leu Leu Ala 420 425 430 Thr GluArg Pro Phe Val Arg Leu Ile Phe Gln Asp Asn Cys Phe Asp 435 440 445 AspAsp Thr Ile Arg Phe Ser Asn Glu Ala 450 455

What is claimed is:
 1. A method for identifying compounds that inhibitNF-kB-dependent transcription or p38-dependent transcription comprising:(a) mixing a test compound with a Pellino-1 polypeptide; (b) assayingthe association of said Pellino-1 polypeptide with a binding partner inthe presence of said test compound; and (c) determining whether the testcompound inhibits the association of said Pellino-1 polypeptide with abinding partner relative to association of said Pellino-1 polypeptidewith said binding partner in the absence of said test compound; whereinsaid Pellino-1 polypeptide comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:4; amino acids ×1 to ×2 of SEQID NO:4, wherein ×1 is any of amino acids 130 through 134 of SEQ IDNO:4, and ×2 is any of amino acids 187 through 191 of SEQ ID NO:4; andamino acids ×1 to ×2 of SEQ ID NO:4, wherein ×1 is any of amino acids 1through 10 of SEQ ID NO:4, and ×2 is any of amino acids 409 through 418of SEQ ID NO:4; and wherein the binding partner is selected from thegroup consisting of IRAK, IRAK-4, and TRAF6.
 2. The method of claim 1,wherein the test compound comprises a molecule selected from the groupconsisting of a small molecule, an antisense oligonucleotide, aribozyme, a double-stranded RNA, a peptide, and an antibody fragment. 3.The method of claim 1, wherein the test compound binds to the Pellino-1polypeptide.
 4. The method of claim 1, wherein the Pellino-1 polypeptidecomprises amino acids 134 through 187 of SEQ ID NO:4.
 5. The method ofclaim 1 wherein the test compound is mixed with the Pellino-1polypeptide in a cell.
 6. The method of claim 5 further comprisingtreating the cell with molecule selected from the group consisting ofinterleukin-1 (IL-1), TNF-alpha, IL-18, phorbol 12-myristate 13-acetate(PMA), peptidoglycan, bacterial lipopeptides, bacteriallipopolysaccharides, zymosan, CpG DNA, flagellin, lipoteichoic acids,and Respiratory Syncytial Virus proteins.
 7. The method of claim 1wherein determining whether the test compound inhibits the associationof said Pellino-1 polypeptide with a binding partner comprisesdetermining the level of NF-kB-dependent transcription.
 8. An inhibitorynucleic acid that binds to a nucleic acid encoding an amino acidsequence selected from the group consisting of: (a) SEQ ID NO:4; (b)amino acids ×1 to ×2 of SEQ ID NO:4, wherein ×1 is any of amino acids130 through 134 of SEQ ID NO:4, and ×2 is any of amino acids 187 through191 of SEQ ID NO:4; and (c) amino acids ×1 to ×2 of SEQ ID NO:4, wherein×1 is any of amino acids 1 through 10 of SEQ ID NO:4, and ×2 is any ofamino acids 409 through 418 of SEQ ID NO:4; wherein the presence of saidinhibitory nucleic acid within a cell inhibits the association ofPellino-1 with a binding partner selected from the group consisting ofIRAK, IRAK-4, and TRAF6.
 9. The inhibitory nucleic acid of claim 8,wherein said inhibitory nucleic acid binds to a nucleic acid comprisinga nucleotide sequence selected from the group consisting of: (a) SEQ IDNO:3; (b) SEQ ID NO:7; (c) SEQ ID NO:11; and (d) an allelic variant of(a)-(c).
 10. The inhibitory nucleic acid of claim 8, wherein saidinhibitory nucleic acid is an antisense oligonucleotide.
 11. Theantisense oligonucleotide of claim 10, wherein said antisenseoligonucleotide is 17 to 30 nucleotides in length.
 12. The inhibitorynucleic acid of claim 8, wherein said inhibitory nucleic acid is aribozyme.
 13. The inhibitory nucleic acid of claim 8, wherein saidinhibitory nucleic acid is a double-stranded RNA.
 14. Thedouble-stranded RNA of claim 13, wherein said double-stranded RNA isformed of RNA strands 19 to 23 nucleotides in length.
 15. The inhibitorynucleic acid of claim 8, wherein the effect of said inhibitory nucleicacid on the association of Pellino-1 with a binding partner is measuredby a method comprising determining the level of NF-kB-dependenttranscription.
 16. An inhibitory polypeptide comprising an antibodyfragment that binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) SEQ ID NO:4; (b) amino acids×1 to ×2 of SEQ ID NO:4, wherein ×1 is any of amino acids 130 through134 of SEQ ID NO:4, and ×2 is any of amino acids 187 through 191 of SEQID NO:4; and (c) amino acids ×1 to ×2 of SEQ ID NO:4, wherein ×1 is anyof amino acids 1 through 10 of SEQ ID NO:4, and ×2 is any of amino acids409 through 418 of SEQ ID NO:4; wherein the presence of said inhibitorypolypeptide within a cell inhibits the association of Pellino-1 with abinding partner selected from the group consisting of IRAK, IRAK-4, andTRAF6.
 17. The inhibitory polypeptide of claim 16 wherein the antibodyfragment is a portion of a human antibody.
 18. The inhibitorypolypeptide of claim 16 wherein the antibody fragment is an Fv fragment.19. The inhibitory polypeptide of claim 16, wherein the antibodyfragment binds to a Pellino-1 polypeptide comprising amino acids 134through 187 of SEQ ID NO:4.
 20. The inhibitory polypeptide of claim 16,wherein the effect of said inhibitory nucleic acid on the association ofPellino-1 with a binding partner is measured by a method comprisingdetermining the level of NF-kB-dependent transcription.